Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes

ABSTRACT

This invention discloses a method for preparing a complex comprised of a VEGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound by identifying a VEGF Nucleic Acid Ligand by SELEX methodology and associating the VEGF Nucleic Acid Ligand with a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound. The invention further discloses Complexes comprising one or more VEGF Nucleic Acid Ligands in association with a Non-Immunogenic, High Molecular Weight Compound or Lipophilic Compound. The invention further includes a Lipid construct comprising a VEGF Nucleic Acid Ligand or Complex and methods for making the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 filing of International ApplicationNumber PCT/US97/18944, filed Oct. 17, 1997, published as WO 98/18480 onMay 7, 1998.

FIELD OF THE INVENTION

Described herein are high affinity 2′Fluoro(2′-F)pyrimidine RNA ligandsto vascular endothelial growth factor (VEGF). The method utilized hereinfor identifying such Nucleic Acid Ligands is called SELEX, an acronymfor Systematic Evolution of Ligands by Exponential enrichment. Furtherincluded in this invention is a method for preparing a therapeutic ordiagnostic Complex comprised of a VEGF Nucleic Acid Ligand and aNon-Immunogenic, High Molecular Weight Compound or a Lipophilic Compoundby identifying a VEGF Nucleic Acid Ligand by SELEX methodology andcovalently linking the VEGF Nucleic Acid Ligand with a Non-Immunogenic,High Molecular Weight Compound or a Lipophilic Compound. The inventionfurther includes Complexes comprised of one or more VEGF Nucleic AcidLigands and a Non-Immunogenic, High Molecular Weight Compound or aLipophilic Compound. The invention further relates to improving thePharmacokinetic Properties of a VEGF Nucleic Acid Ligand by covalentlylinking the VEGF Nucleic Acid Ligand with a Non-Immunogenic, HighMolecular Weight Compound or Lipophilic Compound to form a Complex. Theinvention further relates to improving the Pharmacokinetic Properties ofa VEGF Nucleic Acid Ligand by using a Lipid Construct comprising a VEGFNucleic Acid Ligand or a Complex comprising a VEGF Nucleic Acid Ligandand a Non-Immunogenic, High Molecular Weight Compound or LipophilicCompound. This invention further relates to a method for targeting atherapeutic or diagnostic agent to a biological target that isexpressing VEGF by associating the agent with a Complex comprised of aVEGF Nucleic Acid Ligand and a Lipophilic Compound or Non-Immunogenic,High Molecular Weight Compound, wherein the Complex is furtherassociated with a Lipid Construct and the VEGF Nucleic Acid Ligand isfurther associated with the exterior of the Lipid Construct.

BACKGROUND OF THE INVENTION

A. Selex

The dogma for many years was that nucleic acids had primarily aninformational role. Through a method known as Systematic Evolution ofLigands by EXponential enrichment, termed SELEX, it has become clearthat nucleic acids have three dimensional structural diversity notunlike proteins. SELEX is a method for the in vitro evolution of nucleicacid molecules with highly specific binding to target molecules and isdescribed in U.S. patent application Ser. No. 07/536,428, filed Jun. 11,1990, entitled “Systematic Evolution of Ligands by ExponentialEnrichment,” now abandoned, U.S. patent application Ser. No. 07/714,131,filed Jun. 10, 1991, entitled “Nucleic Acid Ligands,” now U.S. Pat. No.5,475,096, U.S. patent application Ser. No. 07/931,473, filed Aug. 17,1992, entitled Method for Indentifying, “Nucleic Acid Ligands,” now U.S.Pat. No. 5,270,163 (see also WO 91/19813), each of which is specificallyincorporated by reference herein. Each of these applications,collectively referred to herein as the SELEX Patent Applications,describes a fundamentally novel method for making a Nucleic Acid Ligandto any desired target molecule. The SELEX process provides a class ofproducts which are referred to as Nucleic Acid Ligands, each ligandhaving a unique sequence, and which has the property of bindingspecifically to a desired target compound or molecule. EachSELEX-identified Nucleic Acid Ligand is a specific ligand of a giventarget compound or molecule. SELEX is based on the unique insight thatNucleic Acids have sufficient capacity for forming a variety of two- andthree-dimensional structures and sufficient chemical versatilityavailable within their monomers to act as ligands (form specific bindingpairs) with virtually any chemical compound, whether monomeric orpolymeric. Molecules of any size or composition can serve as targets.

The SELEX method involves selection from a mixture of candidateoligonucleotides and step-wise iterations of binding, partitioning andamplification, using the same general selection scheme, to achievevirtually any desired criterion of binding affinity and selectivity.Starting from a mixture of Nucleic Acids, preferably comprising asegment of randomized sequence, the SELEX method includes steps ofcontacting the mixture with the target under conditions favorable forbinding, partitioning unbound Nucleic Acids from those Nucleic Acidswhich have bound specifically to target molecules, dissociating theNucleic Acid-target complexes, amplifying the Nucleic Acids dissociatedfrom the Nucleic Acid-target complexes to yield a ligand-enrichedmixture of Nucleic Acids, then reiterating the steps of binding,partitioning, dissociating and amplifying through as many cycles asdesired to yield highly specific high affinity Nucleic Acid Ligands tothe target molecule.

It has been recognized by the present inventors that the SELEX methoddemonstrates that Nucleic Acids as chemical compounds can form a widearray of shapes, sizes and configurations, and are capable of a farbroader repertoire of binding and other functions than those displayedby Nucleic Acids in biological systems.

The present inventors have recognized that SELEX or SELEX-like processescould be used to identify Nucleic Acids which can facilitate any chosenreaction in a manner similar to that in which Nucleic Acid Ligands canbe identified for any given target. In theory, within a CandidateMixture of approximately 10¹³ to 10¹⁸ Nucleic Acids, the presentinventors postulate that at least one Nucleic Acid exists with theappropriate shape to facilitate each of a broad variety of physical andchemical interactions.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, entitled “Method for Selecting Nucleic Acids on theBasis of Structure,” describes the use of SELEX in conjunction with gelelectrophoresis to select Nucleic Acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection ofNucleic Acid Ligands,” describes a SELEX based method for selectingNucleic Acid Ligands containing photoreactive groups capable of bindingand/or photocrosslinking to and/or photoinactivating a target molecule.U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993,entitled “High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” now U.S. Pat. No. 5,580,737, describes amethod for identifying highly specific Nucleic Acid Ligands able todiscriminate between closely related molecules, which can benon-peptidic, termed Counter-SELEX. U.S. patent application Ser. No.08/143,564, filed Oct. 25, 1993, entitled “Systematic Evolution ofLigands by EXponential Enrichment: Solution SELEX,” now U.S. Pat. No.5,567,588, describes a SELEX-based method which achieves highlyefficient partitioning between oligonucleotides having high and lowaffinity for a target molecule.

The SELEX method encompasses the identification of high-affinity NucleicAcid Ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified Nucleic Acid Ligands containingmodified nucleotides are described in U.S. patent application Ser. No.08/117,991, filed Sep. 8, 1993, entitled “High Affinity Nucleic AcidLigands Containing Modified Nucleotides,” now U.S. Pat. No. 5,660,985,that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S.patent application Ser. No. 08/134,028, supra, describes highly specificNucleic Acid Ligands containing one or more nucleotides modified with2′-amino (2′-NH₂), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S.patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled“Novel Method of Preparation of Known and Novel 2′-Modified Nuceosidesby Intramolecular Nucleophilic Displacement,” describes oligonucleotidescontaining various 2′-modified pyrimidines.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. patent application Ser. No. 08/284,063, filed Aug.2, 1994, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Chimeric SELEX,” now U.S. Pat. No. 5,637,459, and U.S.patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled“Systematic Evolution of Ligands by Exponential Enrichment: BlendedSELEX,” respectively. These applications allow the combination of thebroad array of shapes and other properties, and the efficientamplification and replication properties, of oligonucleotides with thedesirable properties of other molecules.

The SELEX method further encompasses combining selected Nucleic AcidLigands with Lipophilic Compounds or Non-Immunogenic, High MolecularWeight Compounds in a diagnostic or therapeutic Complex as described inU.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled“Nucleic Acid Complexes.” VEGF Nucleic Acid Ligands that are associatedwith a Lipophilic Compound, such as diacyl glycerol or dialkyl glycerol,in a diagnostic or therapeutic complex are described in U.S. patentapplication Ser. No. 08/739,109, filed Oct. 25, 1996, entitled “VascularEndothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes.” VEGFNucleic Acid Ligands that are associated with a High Molecular Weight,Non-Immunogenic Compound, such as Polyethylene glycol or a LipophilicCompound, such as Glycerolipid, phospholipid, or glycerol amide lipid,in a diagnostic or therapeutic complex are described in U.S. patentapplication Ser. No. 08/897,351, filed Jul. 21, 1997, entitled “VascularEndothelial Growth Factor (VEGF) Nucleic Acid Complexes.” Each of theabove described patent applications which describe modifications of thebasic SELEX procedure are specifically incorporated by reference hereinin their entirety.

B. Lipid Constructs

Lipid Bilayer Vesicles are closed, fluid-filled microscopic sphereswhich are formed principally from individual molecules having polar(hydrophilic) and non-polar (lipophilic) portions. The hydrophilicportions may comprise phosphato, glycerylphosphato, carboxy, sulfato,amino, hydroxy, choline or other polar groups. Examples of lipophilicgroups are saturated or unsaturated hydrocarbons such as alkyl, alkenylor other lipid groups. Sterols (e.g., cholesterol) and otherpharmaceutically acceptable adjuvants (including anti-oxidants likealpha-tocopherol) may also be included to improve vesicle stability orconfer other desirable characteristics.

Liposomes are a subset of these bilayer vesicles and are comprisedprincipally of phospholipid molecules that contain two hydrophobic tailsconsisting of fatty acid chains. Upon exposure to water, these moleculesspontaneously align to form spherical, bilayer membranes with thelipophilic ends of the molecules in each layer associated in the centerof the membrane and the opposing polar ends forming the respective innerand outer surface of the bilayer membrane(s). Thus, each side of themembrane presents a hydrophilic surface while the interior of themembrane comprises a lipophilic medium. These membranes may be arrangedin a series of concentric, spherical membranes separated by thin strataof water, in a manner not dissimilar to the layers of an onion, aroundan internal aqueous space. These multilamellar vesicles (MLV) can beconverted into small or Unilamellar Vesicles (UV), with the applicationof a shearing force.

The therapeutic use of liposomes includes the delivery of drugs whichare normally toxic in the free form. In the liposomal form, the toxicdrug is occluded, and may be directed away from the tissues sensitive tothe drug and targeted to selected areas. Liposomes can also be usedtherapeutically to release drugs over a prolonged period of time,reducing the frequency of administration. In addition, liposomes canprovide a method for forming aqueous dispersions of hydrophobic oramphiphilic drugs, which are normally unsuitable for intravenousdelivery.

In order for many drugs and imaging agents to have therapeutic ordiagnostic potential, it is necessary for them to be delivered to theproper location in the body, and the liposome can thus be readilyinjected and form the basis for sustained release and drug delivery tospecific cell types, or parts of the body. Several techniques can beemployed to use liposomes to target encapsulated drugs to selected hosttissues, and away from sensitive tissues. These techniques includemanipulating the size of the liposomes, their net surface charge, andtheir route of administration. MLVs, primarily because they arerelatively large, are usually rapidly taken up by thereticuloendothelial system (principally the liver and spleen). UVs, onthe other hand, have been found to exhibit increased circulation times,decreased clearance rates and greater biodistribution relative to MLVs.

Passive delivery of liposomes involves the use of various routes ofadministration, e.g., intravenous, subcutaneous, intramuscular andtopical. Each route produces differences in localization of theliposomes. Two common methods used to direct liposomes actively toselected target areas involve attachment of either antibodies orspecific receptor ligands to the surface of the liposomes. Antibodiesare known to have a high specificity for their corresponding antigen andhave been attached to the surface of liposomes, but the results havebeen less than successful in many instances. Some efforts, however, havebeen successful in targeting liposomes to tumors without the use ofantibodies, see, for example, U.S. Pat. Nos. 5,019,369, 5,441,745, or5,435,989.

An area of development aggressively pursued by researchers is thedelivery of agents not only to a specific cell type but into the cell'scytoplasm and, further yet, into the nucleus. This is particularlyimportant for the delivery of biological agents such as DNA, RNA,ribozymes and proteins. A promising therapeutic pursuit in this areainvolves the use of antisense DNA and RNA oligonucleotides for thetreatment of disease. However, one major problem encountered in theeffective application of antisense technology is that oligonucleotidesin their phosphodiester form are quickly degraded in body fluids and byintracellular and extracellular enzymes, such as endonucleases andexonucleases, before the target cell is reached. Intravenousadministration also results in rapid clearance from the bloodstream bythe kidney, and uptake is insufficient to produce an effectiveintracellular drug concentration. Liposome encapsulation protects theoligonucleotides from the degradative enzymes, increases the circulationhalf-life and increases uptake efficiency as a result of phagocytosis ofthe Liposomes. In this way, oligonucleotides are able to reach theirdesired target and to be delivered to cells in vivo.

A few instances have been reported where researchers have attachedantisense oligonucleotides to Lipophilic Compounds or Non-Immunogenic,High Molecular Weight Compounds. Antisense oligonucleotides, however,are only effective as intracellular agents. Antisenseoligodeoxyribonucleotides targeted to the epidermal growth factor (EGF)receptor have been encapsulated into Liposomes linked to folate via apolyethylene glycol spacer (folate-PEG-Liposomes) and delivered intocultured KB cells via folate receptor-mediated endocytosis (Wang et al.(1995) Proc. Natl. Acad. Sci USA 92:3318-3322). In addition, alkylenediols have been attached to oligonucleotides (Weiss et al., U.S. Pat.No. 5,245,022). Furthermore, a Lipophilic Compound covalently attachedto an antisense oligonucleotide has been demonstrated in the literature(EP 462 145 B1).

Loading of biological agents into liposomes can be accomplished byinclusion in the lipid formulation or loading into preformed liposomes.Passive anchoring of oligopeptide and oligosaccharide ligands to theexternal surface of liposomes has been described (Zalipsky et al. (1997)Bioconjug. Chem. 8:111:118).

C. VEGF

The growth of new blood vessels from existing endothelium (angiogenesis)is tightly controlled in healthy adults by opposing effects of positiveand negative regulators. Under certain pathological conditions,including proliferative retinopathies, rheumatoid arthritis, psoriasisand cancer, positive regulators prevail and angiogenesis contributes todisease progression (reviewed in Folkman (1995) Nature Medicine1:27-31). In cancer, the notion that angiogenesis represents the ratelimiting step of tumor growth and metastasis (Folkman (1971) New Engl.J. Med. 285:1182-1186) is now supported by considerable experimentalevidence (reviewed in Aznavoorian et al. (1993) Cancer 71:1368-1383;Fidler and Ellis (1994) Cell 79:185-188; Folkman (1990) J. Natl. CancerInst. 82:4-6).

The quantity of blood vessels in tumor tissue is a strong negativeprognostic indicator in breast cancer (Weidner et al. (1992) J. Natl.Cancer Inst. 84:1875-1887), prostate cancer (Weidner et al. (1993) Am.J. Pathol. 143:401-409), brain tumors (Li et al.(1994) Lancet344:82-86), and melanoma (Foss et al.(1996) Cancer Res. 56:2900-2903).

A number of angiogenic growth factors have been described to date amongwhich vascular endothelial growth factor (VEGF) appears to play a keyrole as a positive regulator of physiological and pathologicalangiogenesis (reviewed in Brown et al.(1996) Control ofAngiogenesis(Goldberg and Rosen, eds.) Birkhauser, Basel, in press;Thomas (1996) J. Biol. Chem. 271:603-606). VEGF is a secreteddisulfide-linked homodimer that selectively stimulates endothelial cellsto proliferate, migrate, and produce matrix-degrading enzymes (Conn etal.(1990) Proc. Natl. Acad. Sci. USA 87:1323-1327); Ferrara and Henzel(1989) Biochem. Biophys. Res. Commun. 161:851-858); Gospodarowiczetal.(1989) Proc. Natl. Acad. Sci. USA 7311-7315); Pepper et al.(1991)Biochem. Biophys. Res. Commun. 181:902-906; Unemori et al.(1992) J.Cell. Physiol. 153:557-562), all of which are processes required for theformation of new vessels. In addition to being the only knownendothelial cell specific mitogen, VEGF is unique among angiogenicgrowth factors in its ability to induce a transient increase in bloodvessel permeability to macromolecules (hence its original andalternative name, vascular permeability factor, VPF) (Dvorak etal.(1979) J. Immunol. 122:166-174; Senger et al.(1983) Science219:983-985; Senger et al.(1986) Cancer Res. 46:5629-5632). Increasedvascular permeability and the resulting deposition of plasma proteins inthe extravascular space assists the new vessel formation by providing aprovisional matrix for the migration of endothelial cells (Dvorak etal.(1995) Am. J. Pathol. 146:1029-1039). Hyperpermeability is indeed acharacteristic feature of new vessels, including those associated withtumors (Dvorak et al.(1995) Am. J. Pathol. 146:1029-1039). Furthermore,compensatory angiogenesis induced by tissue hypoxia is now known to bemediated by VEGF (Levy et al.(1996) J. Biol. Chem. 2746-2753); Shweikiet al. (1992) Nature 359:843-845).

VEGF occurs in four forms (VEGF-121, VEGF-165, VEGF-189, VEGF-206) as aresult of alternative splicing of the VEGF gene (Houck et al. (1991)Mol. Endocrin. 5:1806-1814; Tischer et al. (1991) J. Biol. Chem.266:11947-11954). The two smaller forms are diffusable while the largertwo forms remain predominantly localized to the cell membrane as aconsequence of their high affinity for heparin. VEGF-165 also binds toheparin and is the most abundant form. VEGF-121, the only form that doesnot bind to heparin, appears to have a lower affinity for the receptors(Gitay-Goren et al. (1996) J. Biol. Chem. 271:5519-5523) as well aslower mitogenic potency (Keyt et al. (1996) J. Biol. Chem.271:7788-7795). The biological effects of VEGF are mediated by twotyrosine kinase receptors (Flt-1 and Flk-1/KDR) whose expression ishighly restricted to cells of endothelial origin (de Vries et al. (1992)Science 255:989-991; Millauer et al. (1993) Cell 72:835-846; Terman etal. (1991) Oncogene 6:519-524). While the expression of both functionalreceptors is required for high affinity binding, the chemotactic andmitogenic signaling in endothelial cells appears to occur primarilythrough the KDR receptor (Park et al. (1994) J. Biol. Chem.269:25646-25654; Seetharam et al. (1995) Oncogene 10:135-147;Waltenberger et al. (1994) J. Biol. Chem. 26988-26995). The importanceof VEGF and VEGF receptors for the development of blood vessels hasrecently been demonstrated in mice lacking a single allele for the VEGFgene (Carmeliet et al. (1996) Nature 380:435-439; Ferrara et al. (1996)Nature 380:439-442) or both alleles of the Flt-1 (Fong et al. (1995)376:66-70) or Flk-1 genes (Shalaby et al. (1995) Nature 376:62-66). Ineach case, distinct abnormalities in vessel formation were observedresulting in embryonic lethality.

VEGF is produced and secreted in varying amounts by virtually all tumorcells (Brown et al. (1997) Regulation of Angiogenesis (Goldberg andRosen, Eds.) Birkhauser, Basel, pp.233-269). Direct evidence that VEGFand its receptors contribute to tumor growth was recently obtained by ademonstration that the growth of human tumor xenografts in nude micecould be inhibited by neutralizing antibodies to VEGF (Kim et al. (1993)Nature 362:841-844), by the expression of dominant-negative VEGFreceptor flk-1 (Millauer et al. (1996) Cancer Res. 56:1615-1620;Millauer et al. (1994) Nature 367:576-579), by low molecular weightinhibitors of Flk-1 tyrosine kinase activity (Strawn et al. (1966)Cancer Res. 56:3540-3545), or by the expression of antisense sequence toVEGF mRNA (Saleh et al. (1996) Cancer Res. 56:393-401). Importantly, theincidence of tumor metastases was also found to be dramatically reducedby VEGF antagonists (Claffey et al. (1996) Cancer Res. 56:172-181).

In addition to their use as anticancer agents, VEGF inhibitors may beuseful in a wide variety of proliferative diseases characterized byexcessive angiogenesis, including psoriasis, ocular disorders, collagenvascular diseases and rheumatoid arthritis. Although most tumor typesare known to produce VEGF, until recently none has been shown to expressfunctional VEGF receptors. It has been shown that Kaposi's Sarcoma (KS)cells not only produce abundant amounts of VEGF but also expressfunctional VEGF receptors and therefore use VEGF for autocrine growth.Kaposi's sarcoma is typically treated with conventional antimetabolicdrugs. However, a major shortcoming of the use of chemotherapy in KSpatients is the accompanying induction of immunosuppression which hasserious consequences in patients whose immune system is alreadycompromised. The need for alternative therapies is especially great inearly stages of the disease where KS lesions begin to appear but thepatients otherwise feel fairly healthy. In this regard, encapsulation ofchemotherapeutic drugs such as daunorubicin into liposomes has recentlyproved to be a promising method of minimizing side effects ofchemotherapy while maintaining anti-tumor efficacy. Drugs with lowtoxicity that selectively target activated cells of endothelial origin,such as the Nucleic Acid Ligand VEGF antagonists described here, wouldbe an enormous asset in the treatment of KS.

Other areas of potential clinical utility for the VEGF Nucleic AcidLigands are ocular disorders characterized by excessive angiogenesis.Examples of such diseases are macular degeneration and diabeticretinopathy. In macular degeneration, progressive choroidal angiogenesisbeneath the macula (a part of the retina responsible for the highestvisual acuity) interferes with vision. In diabetic retinopathy,angiogenesis in the retina interferes with vision. While the initialstimuli that initiate blood vessel growth in macular degeneration anddiabetic retinopathy are not known at present, VEGF appears to be a keyangiogenesis inducer (Lopez, P. F. et al. (1996) Invest. Ophthalmol.Visual Science 37, 855-868; Kliffen, M. et al. (1997) Br. J. Ophthalmol.81, 154-162; Kvanta, A. et al. (1996) Invest. Ophthalmol. Visual Science37, 1929-1934; Paques et al. (1997) Diabetes & Metabolism 23:125-130).Inhibitors of VEGF therefore may be useful in attenuating angiogenesisin macular degeneration.

SUMMARY OF THE INVENTION

Described herein are high affinity 2′ Fluoro (2′-F)-modifiedpyrimidineRNA ligands to vascular endothelial growth factor (VEGF). The methodutilized herein for identifying such nucleic acid ligands is calledSELEX, an acronym for Systematic Evolution of Ligands by Exponentialenrichment. The ligands described herein were selected from an initialpool of about 10¹⁴ RNA molecules randomized at 30 or 40 contiguouspositions. Included herein are the evolved ligands that are shown inTables 2-6. Further included in this invention is a method for preparinga Complex comprised of a VEGF Nucleic Acid Ligand and a Non-Immunogenic,High Molecular Weight Compound or Lipophilic Compound by the methodcomprising identifying a Nucleic Acid Ligand from a Candidate Mixture ofNucleic Acids where the Nucleic Acid is a ligand of VEGF by the methodof (a) contacting the Candidate Mixture of Nucleic Acids with VEGF, (b)partitioning between members of said Candidate Mixture on the basis ofaffinity to VEGF, and c) amplifying the selected molecules to yield amixture of Nucleic Acids enriched for Nucleic Acid sequences with arelatively higher affinity for binding to VEGF, and covalently linkingsaid identified VEGF Nucleic Acid Ligand with a Non-Immunogenic, HighMolecular Weight Compound or a Lipophilic Compound. The inventionfurther comprises a Complex comprised of a VEGF Nucleic Acid Ligand anda Non-Immunogenic, High Molecular Weight Compound or a LipophilicCompound.

The invention further includes a Lipid Construct comprising a VEGFNucleic Acid Ligand or a Complex. The present invention further relatesto a method for preparing a Lipid Construct comprising a Complex whereinthe Complex is comprised of a VEGF Nucleic Acid Ligand and a LipophilicCompound.

In another embodiment, this invention provides a method for improvingthe pharmacokinetic properties of a VEGF Nucleic Acid Ligand bycovalently linking the VEGF Nucleic Acid Ligand with a Non-Immunogenic,High Molecular Weight Compound or Lipophilic Compound to form a Complexand administering the Complex to a patient. The invention furtherrelates to a method for improving the pharmacokinetic properties of aVEGF Nucleic Acid Ligand by further associating the Complex with a LipidConstruct.

It is an object of the present invention to provide Complexes comprisingone or more VEGF Nucleic Acid Ligands in association with one or moreNon-Immunogenic, High Molecular Weight Compounds or Lipophilic Compoundsand methods for producing the same. It is a further object of thepresent invention to provide Lipid Constructs comprising a Complex. Itis a further object of the invention to provide one or more VEGF NucleicAcid Ligands in association with one or more Non-Immunogenic, HighMolecular Weight Compounds or Lipophilic Compounds with improvedPharmacokinetic Properties.

In embodiments of the invention directed to Complexes comprised of aVEGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular WeightCompound, it is preferred that the Non-Immunogenic, High MolecularWeight Compound is Polyalkylene Glycol, more preferably, polyethyleneglycol (PEG). More preferably, the PEG has a molecular weight of about10-80K. Most preferably, the PEG has a molecular weight of about 20-45K.In embodiments of the invention directed to Complexes comprised of aVEGF Nucleic Acid Ligand and a Lipophilic Compound, it is preferred thatthe Lipophilic Compound is a glycerolipid. In the preferred embodimentsof the invention, the Lipid Construct is preferably a Lipid BilayerVesicle and most preferably a Liposome. In the preferred embodiment, theVEGF Nucleic Acid Ligand is identified according to the SELEX method.

In embodiments of the invention directed to Complexes comprising aNon-Immunogenic, High Molecular Weight Compound or Lipophilic Compoundcovalently linked to a VEGF Nucleic Acid Ligand or Ligands, the VEGFNucleic Acid Ligand or Ligands can serve in a targeting capacity.

Additionally, the VEGF Nucleic Acid Ligand can be associated throughCovalent or Non-Covalent Interactions with a Lipid Construct withoutbeing part of a Complex.

Furthermore, in embodiments of the invention directed to LipidConstructs comprising a VEGF Nucleic Acid Ligand or a Non-Immunogenic,High Molecular Weight or Lipophilic Compound/VEGF Nucleic Acid LigandComplex where the Lipid Construct is of a type that has a membranedefining an interior compartment such as a Lipid Bilayer Vesicle, theVEGF Nucleic Acid Ligand or Complex in association with the LipidConstruct may be associated with the membrane of the Lipid Construct orencapsulated within the compartment. In embodiments where the VEGFNucleic Acid Ligand is in association with the membrane, the VEGFNucleic Acid Ligand can associate with the interior-facing orexterior-facing part of the membrane such that the VEGF Nucleic AcidLigand is projecting into or out of the vesicle. In certain embodiments,a VEGF Nucleic Acid Ligand Complex can be passively loaded onto theoutside of a preformed Lipid Construct. In embodiments where the NucleicAcid Ligand is projecting out of the Lipid Construct, the VEGF NucleicAcid Ligand can serve in a targeting capacity.

In embodiments where the VEGF Nucleic Acid Ligand of the Lipid Constructserves in a targeting capacity, the Lipid Construct can have associatedwith it additional therapeutic or diagnostic agents. In one embodiment,the therapeutic or diagnostic agent is associated with the exterior ofthe Lipid Construct. In other embodiments, the therapeutic or diagnosticagent is encapsulated in the Lipid Construct or associated with theinterior of the Lipid Construct. In yet a further embodiment, thetherapeutic or diagnostic agent is associated with the Complex. In oneembodiment, the therapeutic agent is a drug. In an alternativeembodiment, the therapeutic or diagnostic agent is one or moreadditional Nucleic Acid Ligands.

It is a further object of the present invention to provide a method forinhibiting angiogenesis by the administration of a VEGF Nucleic AcidLigand or a Complex comprising a VEGF Nucleic Acid Ligand andNon-Immunogenic, High Molecular Weight Compound or Lipophilic Compoundor a Lipid Construct comprising the Complex of the present invention. Itis yet a further object of the present invention to provide a method forinhibiting the growth of tumors by the administration of a VEGF NucleicAcid Ligand or Complex comprising a VEGF Nucleic Acid Ligand andNon-Immunogenic, High Molecular Weight Compound or Lipophilic Compoundor a Lipid Construct comprising a Complex of the present invention. Itis yet a further object of the invention to provide a method forinhibiting Kaposi's Sarcoma by the administration of a VEGF Nucleic AcidLigand or Complex comprising a VEGF Nucleic Acid Ligand andNon-Immunogenic, High Molecular Weight Compound or Lipophilic Compoundor a Lipid Construct comprising a Complex of the present invention. Itis yet a further object of the invention to provide a method forinhibiting macular degeneration by the administration of a VEGF NucleicAcid Ligand or Complex comprising a VEGF Nucleic Acid Ligand andNon-Immunogenic, High Molecular Weight Compound or Lipophilic Compoundor a Lipid Construct comprising a Complex of the present invention. Itis yet a further object of the invention to provide a method forinhibiting diabetic retinopathy by the administration of a VEGF NucleicAcid Ligand or Complex comprising a VEGF Nucleic Acid Ligand andNon-Immunogenic, High Molecular Weight Compound or Lipophilic Compoundor a Lipid Construct comprising a Complex of the present invention.

It is a further object of the invention to provide a method fortargeting a therapeutic or diagnostic agent to a biological target thatis expressing VEGF by associating the agent with a Complex comprised ofa VEGF Nucleic Acid Ligand and a Lipophilic Compound or Non-Immunogenic,High Molecular Weight Compound, wherein the Complex is furtherassociated with a Lipid Construct and the VEGF Nucleic Acid Ligand isfurther associated with the exterior of the Lipid Construct.

These and other objects, as well as the nature, scope and utilization ofthis invention, will become readily apparent to those skilled in the artfrom the following description and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1R show the molecular descriptions of NX213 (FIG. 1A), NX278(FIG. 1B), scNX278 (FIG. 1C), scNX213 (FIG. 1D), NX31838-PL(FIG. 1E),NX31838 Lipid Amide 1 (FIG. 1F), NX31838 Lipid Amide 2 (FIG. 1G),NX31838-40K PEG (FIG. 1H), NX31838-20K PEG (FIG. 11), NX31838 40K PEGdimer with no linker (NX31838d0) (FIG. 1J), NX31838 40K dimer with oneC5 linker (NX31838d1) (FIG. 1K), NX31838 40K PEG dimer with two C5linkers (NX31838d2) (FIG. 1L), C-5 Aminolinker (FIG. 1M), GlycerolBisphosphate Linker (FIG. 1N), 18 Atom Spacer Linker (FIG. 1O),Aminotetraethylene Glycol Linker (FIG. 1P), 3′3′ dT (FIG. 1Q), andNX31917 (FIG. 1R). The 5′ phosphate group of the ligand is depicted inthe figures, mPEG stands for methyl polyethylene glycol. A lower caseletter preceding a nucleotide indicates the following: m=2′-O-Methyl,a=2′-amino, r=ribo, and f=2′-fluoro. No letter preceding a nucleotideindicates a deoxyribonucleotide(2′H). 3′3′-dT indicates a 3′3′ invertedphosphodiesterlinkage at the 3′ end. An S following a nucleotide denotesa backbone modification consisting of a phosphorothioate internucleosidelinkage.

FIG. 2 shows binding properties of various Nucleic Acid Ligands to VEGF.The binding affinities of the unmodified Nucleic Acid Ligand (NX213,open circle), its dialkyl glycerol modified analog (NX278, open diamond)and liposomal NX278 (NX278-L, open square), along with the sequencescrambled (sc) controls (scNX213, closed circle; scNX278, closeddiamond; and scNX278-L, closed square) were determined by a competitionelectrophoreticmobility shift assay. NX213 is 5′-TsTsTsTs mAaCaC aCaUrGrAaUmG rGaUmA mGrAaC mGaCaC mGmGmG mGaUmG TsTsTsTsT-3′ and scNX213 is(SEQ ID NO:1) 5′-TsTsTsTs mGaUaC mGmGaU mAaCrG mGrAmG aUmGrG rAaCnCmGaUaC mAaCmG TsTsTsTsT-3′ (SEQ ID NO:4) ³²P 5=end-labeled NX-213 (1.5nM) was incubated in binding buffer (phosphate buffered saline with0.01% human serum albumin) at 37° C. for 20 min in the presence of VEGF(0.33 nM) and competitor oligonucleotide(5 pM-0.33 μM). The ³²pNX-213/VEGF complex was resolved from the free ³²p NX-213 byelectrophoresison 8% polyacrylamide gel (19:1 acrylamide:bis-acrylamide,Tris-borate, 89 mM, 1 mM EDTA as the running buffer). The intensity ofthe band corresponding to ³²P NX-213/VEGF complex at varying competitorconcentrations was quantitated by phosphorimager analysis. Datanormalized for the amount of complex formed in the absence of competitorwere fitted by the least squares method to the competition bindingequation.

FIG. 3 shows the effect of various Nucleic Acid Ligands on VEGF-inducedincreases in vascular permeability. VEGF (20 nM) with or without NucleicAcid Ligands was injected intradermally to guinea pigs that hadpreviously received an injection of Evans blue dye. The amount of dyeleakage was quantitated by measuring the relative amount of lightabsorbed by the skin at the site of injection.

FIG. 4 shows that NX278-L inhibits KS cell growth. Growth of KSY-1 cellsin the presence of various concentrations of NX213, NX278-L and scNX278-L. KSY-1 cells were seeded in 24 well plates at a density of 1×10⁴cells/well on day 0. Fresh medium treated identically was replaced ondays 1 and 3. The cell numbers were determined by trypsinization ofcells on day 5 or 6 of culture using particle coulter counter. Theexperiments were done in triplicates several times. Results shown arethe average and SE of representative experiment.

FIGS. 5A and 5B show that NX278 inhibits KS cell growth in athymic mice.Athymic mice were implanted with KS tumor behind the forelegs on day 1.Mice were treated with NX278-L (50 μg/day/mouse, FIG. 5A and 150μg/day/mouse, FIG. 5B) by intraperitoneal injection daily for five daysbeginning on day 2. Control mice were treated with empty liposomes usingthe same quantity of lipids as the Nucleic Acid Ligand treated group.The tumor sizes were measured over the period of two weeks. The tumorswere removed on day 14 and measured.

FIG. 6 summarizes the data for the plasma concentration of NX31838 20KPEG (□), 40K PEG (▪), and NX31838 (minus PEG) (∇) as a function of timefollowing the bolus injection.

FIG. 7 summarizes the data for the plasma concentration of NX31838 PL asa function of time following the bolus injection.

FIGS. 8A-8D show changes in vascular permeability elicited byintradermal injection of VEGF protein (0.8 pmol)±Nucleic AcidLigand/monoclonal antibody as indicated. Local extravasation of Evansblue dye was determined 30 min after injection by transillumination ofharvested skin. FIGS. A, B, C, and D show the effect of co-mixingNX31838-20K PEG, NX31838-40K PEG, NX31838-PL, or NX31838d2-40K PEG withprotein 30 min prior to injection. Values are mean±SEM. *P<0.05 comparedwith VEGF alone. See FIG. 1 for molecular descriptions.

FIGS. 9A-9C show the evaluation of Nucleic Acid Ligand attenuation ofVEGF-induced corneal angiogenesis. Zero or three pmol of VEGF proteinwere incorporated in a biopolymer (Hydron) and implanted in the cornealstroma. Animals were treated intravenously twice daily with either PBSor Nucleic Acid Ligand as indicated for 5 days. FIGS. A, B, and Cillustrate the effect of systemic treatment with NX31838-20K PEG,NX31838-40K PEG, or NX31838-PL Nucleic Acid Ligand onneovascularization. Values are mean±SEM. * P<0.05 compared with 3 pmolVEGF+PBS group. See FIG. 1 for molecular descriptions.

FIG. 10 summarizes the data for the plasma (∘,Δ) or vitreous (,▴,▪)concentration of NX31838-40K PEG as a function of time followingadministration.

FIG. 11 shows tumor growth curves of human A673 tumors growingsubcutaneously (s.c.) in nude mice treated with 40 mg/kg or 10 mg/kg ofVEGF NX31838 40K PEG Nucleic Acid Ligand (NX 31838 NAL) delivered twicea day (BID). A negative control consisted of a scrambled VEGF NucleicAcid Ligand sequence, NX31917 NAL (see FIG. 1R for moleculardescription), dosed at 40 mg/kg twice daily, and a positive controlconsisted of an anti-VEGF monoclonal antibody mAb 26503.11 (R&D Systems)dosed at 100 μg/mouse twice weekly. Since there appeared to be nosignificant difference between the 40 mg/kg dose group and the 10 mg/kgdose group, no further dosing of the 40 mg/kg group occurred after day14. Groups of 8 mice were implanted s.c. with 1×10⁷ A673 tumor cells onday 0, and treatment with test compounds by intraperitoneal injectionsinitiated on day 1 for the duration of the experiment. Tumor volume,expressed as mm³, was determined using the formula: Tumor vol.=L×W²/2.

FIG. 12 shows tumor growth curves of different dose schedules(comparison of twice daily dosing (BID) to once daily dosing (QD)), 40KPEG batches (comparison NX31838.07 batch with the new NX31838.04 batch),and different drug formulations (comparison of liposomal VEGF NX31838PLNAL to VEGF NX31838 NAL 40K PEG) of VEGF NX31838 Nucleic Acid Ligand(NAL). Groups of 8 mice were implanted s.c. with 1×10⁷ A673 tumor cellson day 0, and treatment with test compounds by intraperitonealinjections initiated on day 1 for the duration of the experiment.Several groups had animals where the tumors failed to grow, andconsequently for final analysis some groups contain only 7 (NX31838.0410 mg/kg BID, and NX31838.04 3 mg/kg BID), or 6 (NX31838.04 10 mg/kg QD,and NX31838.07 10 mg/kg BID) animals. Tumor volume, expressed as mm³,was determined using the formula: Tumor vol.=L×W²/2.

FIG. 13 shows dose-dependent inhibition of A673 tumors growingsubcutaneously (s.c.) in nude mice by VEGF NX31838 40K PEG Nucleic AcidLigand (NX31838 NAL) delivered once daily. This titration failed toreach a no effect dose; tumor inhibition was still observed with thelowest (0.03 mg/kg) dose. Groups of 8 mice were implanted s.c. with1×10⁷ A673 tumor cells on day 0, and treatment with test compounds byintraperitoneal injections initiated on day 1 for the duration of theexperiment; group NX31838 NAL 3 mg/kg had 2 animals where tumors failedto grow and consequently contains only 6 animals. Tumor volume,expressed as mm³, was determined using the formula: Tumor vol.=L×W²/2.

FIG. 14 shows tumor growth curves demonstrating inhibition of staged(i.e., established) A673 tumors growing subcutaneously (s.c.) in nudemice by VEGF NX31838 40K PEG Nucleic Acid Ligand (NAL) delivered oncedaily. A positive control consisted of an anti-VEGF monoclonal antibodymAb 26503.11 (R&D Systems) dosed at 100 μg/mouse twice weekly. Mice wereimplanted with 1×10⁷ A673 cells, and tumors allowed to grow to a volumeof 200±100 mm³, at which time animals were sorted by weight, tattooedfor permanent identification, and treatment with test compounds byintraperitoneal injections initiated and continued for the duration ofthe experiment. Each point represents the mean of 8 mice. Tumor volume,expressed as mm³, was determined using the formula: Tumor vol.=L×W²/2.

FIG. 15 summarizes the data for the plasma concentration of NX213,NX278, NX278-Liposome following bolus injection.

FIG. 16 shows the growth curves of KSY-1 tumors implanted subcutaneouslyin nude mice. The mice were treated by intraperitoneal injections ofNX31917 40K PEG or is NX31838 40K PEG (30 mg/kg) or PBS twice daily forthe duration of the experiment. Treatment was initiated one day aftersubcutaneous implantation of 2×10⁷ KSY-1 cells in the hind flank of nudemice. Four mice were used in each group. Errors are SEM.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

“Covalent Bond” is the chemical bond formed by the sharing of electrons.

“Non-Covalent Interactions” are means by which molecular entities areheld together by interactions other than Covalent Bonds including ionicinteractions and hydrogen bonds.

“Lipophilic Compounds” are compounds which have the propensity toassociate with or partition into lipid and/or other materials or phaseswith low dielectric constants, including structures that are comprisedsubstantially of lipophilic components. Lipophilic Compounds includelipids as well as non-lipid containing compounds that have thepropensity to associate with lipid (and/or other materials or phaseswith low dielectric constants). Cholesterol, phospholipid,andglycerolipids, such as dialkylglycerol, and diacylglycerol, and glycerolamide lipids are further examples of Lipophilic Compounds. In onepreferred embodiment of the invention, the lipophilic compoundcovalently linked to the VEGF Nucleic Acid Ligand is a glycerolipidhaving the structure

where R¹, R², and R³ are independently selected from the groupconsisting of CH₃(CH)_(n)—O(PO₃)—CH₂—; and CH₃(CH₂)_(n)—CONH₂—CH₂—,CH₃(CH₂)_(n)O—, CH₃(CH₂)_(n)OCH₂—, CH₃(CH₂)_(n)(CO)OCH₂—,CH₃(CH₂)_(n)(CO)O— and X—, wherein at least one must be X—, and X isindependently selected from the group consisting of (PO₄), O andCH₂OC═O, and wherein n=0-30, preferably 10-20. When R isCH₃(CH₂)_(n)—O(PO₃)—CH₂—, the Lipophilic Compound is a phospholipid.When R is CH₃(CH₂)_(n)—CONH₂—CH₂—, the Lipophilic Compound is a glycerolamide lipid. When R is CH₃(CH₂)_(n)O— or CH₃(CH₂)_(n)OCH₂—, theLipophilic Compound is a dialkylglycerollipid. When R isCH₃(CH₂)_(n)(CO)OCH₂— or CH₃(CH₂)_(n)(CO)O—; the Lipophilic Compound isdiacylglycerol lipid. In a preferred embodiment, R³ is X—.

“Complex” as used herein describes the molecular entity formed by thecovalent linking of a VEGF Nucleic Acid Ligand to a Non-Immunogenic,High Molecular Weight Compound or Lipophilic Compound. In certainembodiments of the present invention, the Complex is depicted as A-B-Y,wherein A is a Lipophilic Compound or Non-Immunogenic, High MolecularWeight Compound as described herein; B is optional, and may be one ormore linkers Z; and Y is a VEGF Nucleic Acid Ligand.

“Lipid Constructs,” for purposes of this invention, are structurescontaining lipids, phospholipids, or derivatives thereof comprising avariety of different structural arrangements which lipids are known toadopt in aqueous suspension. These structures include, but are notlimited to, Lipid Bilayer Vesicles, micelles, Liposomes, emulsions,lipid ribbons or sheets, and may be complexed with a variety of drugsand components which are known to be pharmaceutically acceptable. In thepreferred embodiment, the Lipid Construct is a Liposome. The preferredLiposome is unilarnellarand has a relative size less than 200 nm. Commonadditional components in Lipid Constructs include cholesterol andalpha-tocopherol, among others. The Lipid Constructs may be used aloneor in any combination which one skilled in the art would appreciate toprovide the characteristics desired for a particular application. Inaddition, the technical aspects of Lipid Constructs and Liposomeformation are well known in the art and any of the methods commonlypracticed in the field may be used for the present invention.

“Nucleic Acid Ligand” as used herein is a non-naturally occurringNucleic Acid having a desirable action on a Target. The Target of thepresent invention is VEGF, hence the term VEGF Nucleic Acid Ligand. Adesirable action includes, but is not limited to, binding of the Target,catalytically changing the Target, reacting with the Target in a waywhich modifies/alters the Target or the functional activity of theTarget, covalently attaching to the Target as in a suicide inhibitor,facilitating the reaction between the Target and another molecule. Inthe preferred embodiment, the action is specific binding affinity forVEGF, wherein the Nucleic Acid Ligand is not a Nucleic Acid having theknown physiological function of being bound by VEGF.

In preferred embodiments of the invention, the VEGF Nucleic Acid Ligandof the Complexes and Lipid Constructs of the invention are identified bythe SELEX methodology. VEGF Nucleic Acid Ligands are identified from aCandidate Mixture of Nucleic Acids, said Nucleic Acid being a ligand ofVEGF, by the method comprising a) contacting the Candidate Mixture withVEGF, wherein Nucleic Acids having an increased affinity to VEGFrelative to the Candidate Mixture may be partitioned from the remainderof the Candidate Mixture; b) partitioning the increased affinity NucleicAcids from the remainder of the Candidate Mixture; and c) amplifying theincreased affinity Nucleic Acids to yield a ligand-enriched mixture ofNucleic Acids (see U.S. patent application Ser. No. 08/233,012, filedApr. 25, 1994, entitled “High Affinity Oligonucleotides to VascularEndothelial Growth Factor (VEGF),” U.S. patent application Ser. No.08/447,169, filed May 19, 1995, entitled “High Affinity OligonucleotideLigands to Vascular Endothelial Growth Factor (VEGF),” which are herebyincorporated by reference herein).

“Candidate Mixture” is a mixture of Nucleic Acids of differing sequencefrom which to select a desired ligand. The source of a Candidate Mixturecan be from naturally-occuring Nucleic Acids or fragments thereof,chemically synthesized Nucleic Acids, enzymatically synthesized NucleicAcids or Nucleic Acids made by a combination of the foregoingtechniques. In a preferred embodiment, each Nucleic Acid has fixedsequences surrounding a randomized region to facilitate theamplification process.

“Nucleic Acid” means either DNA, RNA, single-stranded or double-strandedand any chemical modifications thereof. Modifications include, but arenot limited to, those which provide other chemical groups thatincorporate additional charge, polarizability, hydrogen bonding,electrostatic interaction, and fluxionality to the Nucleic Acid Ligandbases or to the Nucleic Acid Ligand as a whole. Such modificationsinclude, but are not limited to, 2′-position sugar modifications,5-position pyrimidine modifications, 8-position purine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodo-uracil, backbone modifications such asinternucleoside phosphorothioate linkages, methylations, unusualbase-pairing combinations such as the isobases isocytidine andisoguanidine and the like. Modifications can also include 3′ and 5′modifications such as capping.

“Non-Immunogenic, High Molecular Weight Compound” is a compound betweenapproximately 1000 Da to 1,000,000 Da, more preferably approximately1000 Da to 500,000 Da, and most preferably approximately 1000 Da to200,000 Da, that typically does not generate an immunogenic response.For the purposes of this invention, an immunogenic response is one thatcauses the organism to make antibody proteins. Examples ofNon-Immunogenic, High Molecular Weight Compounds include PolyalkyleneGlycol and polyethylene glycol. In one preferred embodiment of theinvention, the Non-Immunogenic, High Molecular Weight Compoundcovalently linked to the VEGF Nucleic Acid Ligand is a polyalkyleneglycol and has the structure R(O(CH₂)_(x))_(n)O—, where R isindependently selected from the group consisting of H and CH₃, x=2-5,and n≈MW of the Polyalkylene Glycol/16+14x. In the preferred embodimentof the present invention, the molecular weight is about between 10-80kDa. In the most preferred embodiment, the molecular weight of thepolyalkylene glycol is about between 20-45 kDa. In the most preferredembodiment, x=2 and n=9×10². There can be one or more PolyalkyleneGlycols attached to the same VEGF Nucleic Acid Ligand, with the sum ofthe molecular weights preferably being between 10-80 kDa, morepreferably 20-45 kDa. In certain embodiments, the Non-Immunogenic, HighMolecular Weight Compound can also be a Nucleic Acid Ligand.

“Lipid Bilayer Vesicles” are closed, fluid-filled microscopic sphereswhich are formed principally from individual molecules having polar(hydrophilic) and non-polar (lipophilic) portions. The hydrophilicportions may comprise phosphato, glycerylphosphato,carboxy, sulfato,amino, hydroxy, choline and other polar groups. Examples of non-polargroups are saturated or unsaturated hydrocarbons such as alkyl, alkenylor other lipid groups. Sterols (e.g., cholesterol) and otherpharmaceutically acceptable components (including anti-oxidants likealpha-tocopherol) may also be included to improve vesicle stability orconfer other desirable characteristics.

“Liposomes” are a subset of Lipid Bilayer Vesicles and are comprisedprincipally of phospholipid molecules which contain two hydrophobictails consisting of long fatty acid chains. Upon exposure to water,these molecules spontaneously align to form a bilayer membrane with thelipophilic ends of the molecules in each layer associated in the centerof the membrane and the opposing polar ends forming the respective innerand outer surface of the bilayer membrane. Thus, each side of themembrane presents a hydrophilic surface while the interior of themembrane comprises a lipophilic medium. These membranes when formed aregenerally arranged in a system of concentric closed membranes separatedby interlamellar aqueous phases, in a manner not dissimilar to thelayers of an onion, around an internal aqueous space. Thesemultilamellar vesicles (MLV) can be converted into unilamellar vesicles(UV), with the application of a shearing force.

“Cationic Liposome” is a Liposome that contains lipid components thathave an overall positive charge at physiological pH.

“SELEX” methodology involves the combination of selection of NucleicAcid Ligands which interact with a Target in a desirable manner, forexample binding to a protein, with amplification of those selectedNucleic Acids. Iterative cycling of the selection/amplification stepsallows selection of one or a small number of Nucleic Acids whichinteract most strongly with the Target from a pool which contains a verylarge number of Nucleic Acids. Cycling of the selection/amplificationprocedure is continued until a selected goal is achieved. The SELEXmethodology is described in the SELEX Patent Applications.

“Target” means any compound or molecule of interest for which a ligandis desired. A Target can be a protein (such as VEGF, thrombin, andselectin), peptide, carbohydrate, polysaccharide, glycoprotein, hormone,receptor, antigen, antibody, virus, substrate, metabolite, transitionstate analog, cofactor, inhibitor, drug, dye, nutrient, growth factor,etc. without limitation. The principal Target of the subject inventionis VEGF.

“Improved Pharmacokinetic Properties” means that the VEGF Nucleic AcidLigand covalently linked to a Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound or in association with a Lipid Constructshows a longer circulation half-life in vivo relative to the same VEGFNucleic Acid Ligand not in association with a Non-Immunogenic, HighMolecular Weight Compound or Lipophilic Compound or in association witha Lipid Construct.

“Linker” is a molecular entity that connects two or more molecularentities through Covalent Bond or Non-Covalent Interactions, and canallow spatial separation of the molecular entities in a manner thatpreserves the functional properties of one or more of the molecularentities. A linker can also be known as a spacer. Examples of Linkers,include but are not limited to, the structures shown in FIGS. 1M-1P.

“Therapeutic” as used herein, includes treatment and/or prophylaxis.When used, Therapeutic refers to humans and other animals.

This invention includes RNA ligands to VEGF that are comprised of2′F-modified nucleotides. This invention further includes the specificRNA ligands to VEGF shown in Tables 2-6 (SEQ ID NOS:15-132). Morespecifically, this invention includes nucleic acid sequences that aresubstantially homologous to and that have substantially the same abilityto bind VEGF as the specific nucleic acid ligands shown in Tables 2-6.By substantially homologous it is meant a degree of primary sequencehomology in excess of 70%, most preferably in excess of 80%, and evenmore preferably in excess of 90%, 95%, or 99%. The percentage ofhomology as described herein is calculated as the percentage ofnucleotides found in the smaller of the two sequences which align withidentical nucleotide residues in the sequence being compared when 1 gapin a length of 10 nucleotides may be introduced to assist in thatalignment. Substantially the same ability to bind VEGF means that theaffinity is within one or two orders of magnitude of the affinity of theligands described herein. It is well within the skill of those ofordinary skill in the art to determine whether a givensequence—substantially homologous to those specifically describedherein—has the same ability to bind VEGF.

A review of the sequence homologies of the nucleic acid ligands of VEGFshown in Tables 2-6 (SEQ ID NOS:15-132) shows that sequences with littleor no primary homology may have substantially the same ability to bindVEGF. For these reasons, this invention also includes Nucleic AcidLigands that have substantially the same postulated structure orstructural motifs and ability to bind VEGF as the nucleic acid ligandsshown in Tables 2-6. Substantially the same structure or structuralmotifs can be postulated by sequence alignment using the Zukerfoldprogram (see Zuker (1989) Science 244:48-52). As would be known in theart, other computer programs can be used for predicting secondarystructure and strutural motifs. Substantially the same structure orstructural motif of Nucleic Acid Ligands in solution or as a boundstructure can also be postulated using NMR or other techniques as wouldbe known in the art.

Further included in this invention is a method for preparing a Complexcomprised of a VEGF Nucleic Acid Ligand and a Non-Immunogenic, HighMolecular Weight Compound or Lipophilic Compound by the methodcomprising identifying a Nucleic Acid Ligand from a Candidate Mixture ofNucleic Acids where the Nucleic Acid is a ligand of VEGF by the methodof (a) contacting the Candidate Mixture of Nucleic Acids with VEGF, (b)partitioning between members of said Candidate Mixture on the basis ofaffinity to VEGF, and c) amplifying the selected molecules to yield amixture of Nucleic Acids enriched for Nucleic Acid sequences with arelatively higher affinity for binding to VEGF, and covalently linkingsaid identified VEGF Nucleic Acid Ligand with a Non-Immunogenic, HighMolecular Weight Compound or a Lipophilic Compound.

It is a further object of the present invention to provide Complexescomprising one or more VEGF Nucleic Acid Ligands covalently linked to aNon-Immunogenic, High Molecular Weight Compound or Lipophilic Compound.Such Complexes have one or more of the following advantages over a VEGFNucleic Acid Ligand not in association with a Non-Immunogenic, HighMolecular Weight Compound or Lipophilic Compound: 1) ImprovedPharmacokinetic Properties, and 2) improved capacity for intracellulardelivery, or 3) improved capacity for targeting. Complexes furtherassociated with a Lipid Construct have the same advantages.

The Complexes or the Lipid Constructs comprising the VEGF Nucleic AcidLigand or Complexes may benefit from one, two, or three of theseadvantages. For example, a Lipid Construct of the present invention maybe comprised of a) a Liposome, b) a drug that is encapsulated within theinterior of the Liposome, and c) a Complex comprised of a VEGF NucleicAcid Ligand and Lipophilic Compound, wherein the VEGF Nucleic AcidLigand component of the Complex is associated with and projecting fromthe exterior of the Lipid Construct. In such a case, the Lipid Constructcomprising a Complex will 1) have Improved Pharmacokinetic Properties,2) have enhanced capacity for intracellular delivery of the encapsulateddrug, and 3) be specifically targeted to the preselected location invivo that is expressing VEGF by the exteriorly associated VEGF NucleicAcid Ligand.

In another embodiment, this invention provides a method for improvingthe pharmacokinetic properties of a VEGF Nucleic Acid Ligand bycovalently linking the VEGF Nucleic Acid Ligand with a Non-Immunogenic,High Molecular Weight Compound or Lipophilic Compound to form a Complexand administering the Complex to a patient. The invention furtherrelates to a method for improving the pharmacokinetic properties of aVEGF Nucleic Acid Ligand by further associating the Complex with a LipidConstruct.

In another embodiment, the Complex of the present invention is comprisedof a VEGF Nucleic Acid Ligand covalently attached to a LipophilicCompound, such as a glycerolipid, or a Non-Immunogenic, High MolecularWeight Compound, such as Polyalkylene Glycol or polyethylene glycol(PEG). In these cases, the pharmacokinetic properties of the Complexwill be enhanced relative to the VEGF Nucleic Acid Ligand alone. Inanother embodiment, the pharmacokinetic properties of the VEGF NucleicAcid Ligand is enhanced relative to the VEGF Nucleic Acid Ligand alonewhen the VEGF Nucleic Acid Ligand is covalently attached to aNon-Immunogenic, High Molecular Weight Compound or Lipophilic Compoundand is further associated with a Lipid Construct or the VEGF NucleicAcid Ligand is encapsulated within a Lipid Construct.

In embodiments where there are multiple VEGF Nucleic Acid Ligands, thereis an increase in avidity due to multiple binding interactions withVEGF. Furthermore, in embodiments where the Complex is comprised ofmultiple VEGF Nucleic Acid Ligands, the pharmacokinetic properties ofthe Complex will be improved relative to one VEGF Nucleic Acid Ligandalone. In embodiments where a Lipid Construct comprises multiple NucleicAcid Ligands or Complexes, the Pharmacokinetic Properties of the VEGFNucleic Acid Ligand may be improved relative to Lipid Constructs inwhich there is only one Nucleic Acid Ligand or Complex.

In certain embodiments of the invention, the Complex of the presentinvention is comprised of a VEGF Nucleic Acid Ligand attached to one(dimeric) or more (multimeric) other Nucleic Acid Ligands. The NucleicAcid Ligand can be to VEGF or a different Target. In embodiments wherethere are multiple VEGF Nucleic Acid Ligands, there is an increase inavidity due to multiple binding interactions with VEGF. Furthermore, inembodiments of the invention where the Complex is comprised of a VEGFNucleic Acid Ligand attached to one or more other VEGF Nucleic AcidLigands, the pharmacokinetic properties of the Complex will be improvedrelative to one VEGF Nucleic Acid Ligand alone.

The Non-Immunogenic, High Molecular Weight compound or LipophilicCompound may be covalently bound to a variety of positions on the VEGFNucleic Acid Ligand, such as to an exocyclic amino group on the base,the 5-position of a pyrimidine nucleotide, the 8-position of a purinenucleotide, the hydroxyl group of the phosphate, or a hydroxyl group orother group at the 5′ or 3′ terminus of the VEGF Nucleic Acid Ligand. Inembodiments where the Lipophilic Compound is a glycerolipid, or theNon-Immunogenic, High Molecular Weight Compound is polyalkylene glycolor polyethylene glycol, preferably it is bonded to the 5′ or 3′ hydroxylof the phosphate group thereof. In the most preferred embodiment, theLipophilic Compound or Non-Immunogenic, High Molecular Weight Compoundis bonded to the 5′ hydroxyl of the phosphate group of the Nucleic AcidLigand. Attachment of the Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound to the VEGF Nucleic Acid Ligand can bedone directly or with the utilization of Linkers or Spacers. Inembodiments where the Lipid Construct comprises a Complex, or where theVEGF Nucleic Acid Ligands are encapsulated within the Liposome, aNon-Covalent Interaction between the VEGF Nucleic Acid Ligand or theComplex and the Lipid Construct is preferred.

One problem encountered in the therapeutic use of Nucleic Acids is thatoligonucleotides in their phosphodiester form may be quickly degraded inbody fluids by intracellular and extracellular enzymes such asendonucleases and exonucleases before the desired effect is manifest.Certain chemical modifications of the VEGF Nucleic Acid Ligand can bemade to increase the in vivo stability of the VEGF Nucleic Acid Ligandor to enhance or to mediate the delivery of the VEGF Nucleic AcidLigand. Modifications of the VEGF Nucleic Acid Ligands contemplated inthis invention include, but are not limited to, those which provideother chemical groups that incorporate additional charge,polarizability, hydrophobicity, hydrogen bonding, electrostaticinteraction, and fluxionality to the VEGF Nucleic Acid Ligand bases orto the VEGF Nucleic Acid Ligand as a whole. Such modifications include,but are not limited to, 2′-position sugar modifications, 5-positionpyrimidine modifications, 8-position purine modifications, modificationsat exocyclic amines, substitution of 4-thiouridine, substitution of5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate oralkyl phosphate modifications, methylations, unusual base-pairingcombinations such as the isobases isocytidine and isoguanidine and thelike. Modifications can also include 3′ and 5′ modifications such ascapping.

Where the Nucleic Acid Ligands are derived by the SELEX method, themodifications can be pre- or post-SELEX modifications. Pre-SELEXmodifications yield VEGF Nucleic Acid Ligands with both specificity forVEGF and improved in vivo stability. Post-SELEX modifications made to2′-OH Nucleic Acid Ligands can result in improved in vivo stabilitywithout adversely affecting the binding capacity of the Nucleic AcidLigands. The preferred modifications of the VEGF Nucleic Acid Ligands ofthe subject invention are 5′ and 3′ phosphorothioate capping and/or 3′3′inverted phosphodiester linkage at the 3′ end. In the most preferredembodiment, the preferred modification of the VEGF Nucleic Acid

Ligand is 3′3′ inverted phosphodiester linkage at the 3′ end. Additional2′ fluoro (2′-F), 2′ amino (2′-NH₂) and 2′ O methyl (2′-OMe)modification of some or all of the nucleotides is preferred.

In another aspect of the present invention, the covalent linking of theVEGF Nucleic Acid Ligand with a Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound results in Improved PharmacokineticProperties (i.e., slower clearance rate) relative to the VEGF NucleicAcid Ligand not in association with a Non-Immunogenic, High MolecularWeight Compound or Lipophilic Compound.

In another aspect of the present invention, the Complex comprising aVEGF Nucleic Acid Ligand and Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound can be further associated with a LipidConstruct. This association may result in Improved PharmacokineticProperties relative to the VEGF Nucleic Acid Ligand or Complex not inassociation with a Lipid Construct. The VEGF Nucleic Acid Ligand orComplex can be associated with the Lipid Construct through covalent orNon-Covalent Interactions. In a preferred embodiment. the association isthrough Non-Covalent Interactions. In a preferred embodiment. the LipidConstruct is a Lipid Bilayer Vesicle. In the most preferred embodiment,the Lipid Construct is a Liposome.

Liposomes for use in the present invention can be prepared by any of thevarious techniques presently known in the art or subsequently developed.Typically, they are prepared from a phospholipid, for example,distearoyl phosphatidylcholine,and may include other materials such asneutral lipids, for example, cholesterol, and also surface modifierssuch as positively charged (e.g., sterylamine or aminomannose oraminomannitol derivatives of cholesterol) or negatively charged (e.g.,diacetyl phosphate, phosphatidyl glycerol) compounds. MultilamellarLiposomes can be formed by conventional techniques, that is, bydepositing a selected lipid on the inside wall of a suitable containeror vessel by dissolving the lipid in an appropriate solvent, and thenevaporating the solvent to leave a thin film on the inside of the vesselor by spray drying. An aqueous phase is then added to the vessel with aswirling or vortexing motion which results in the formation of MILVs.UVs can then be formed by homogenization, sonication or extrusion(through filters) of MLV'S. In addition, UVs can be formed by detergentremoval techniques.

In certain embodiments of this invention. the Lipid Construct comprisesa targeting VEGF Nucleic Acid Ligand(s) associated with the surface ofthe Lipid Construct and an encapsulated therapeutic or diagnostic agent.Preferably the Lipid Construct is a Liposome. Preformed Liposomes can bemodified to associate with the VEGF Nucleic Acid Ligands. For example, aCationic Liposome associates through electrostatic interactions with theVEGF Nucleic Acid Ligand. A VEGF Nucleic Acid Ligand covalently linkedto a Lipophilic Compound, such as a glycerolipid, can be added topreformed Liposomes whereby the glycerolipid, phospholipid, or glycerolamide lipid becomes associated with the liposomal membrane.Alternatively, the VEGF Nucleic Acid Ligand can be associated with theLiposome during the formulation of the Liposome.

It is well known in the art that Liposomes are advantageous forencapsulating or incorporating a wide variety of therapeutic anddiagnostic agents. Any variety of compounds can be enclosed in theinternal aqueous compartment of the Liposomes. Illustrative therapeuticagents include antibiotics, antiviral nucleosides, antifungalnucleosides, metabolic regulators, immune modulators, chemotherapeuticdrugs, toxin antidotes, DNA, RNA, antisense oligonucleotides, etc. Bythe same token, the Lipid Bilayer Vesicles may be loaded with adiagnostic radionuclide (e.g., Indium 111, Iodine 131, Yttrium 90,Phosphorous 32, or gadolinium) and fluorescent materials or othermaterials that are detectable in in vitro and in vivo applications. Itis to be understood that the therapeutic or diagnostic agent can beencapsulated by the Liposome walls in the aqueous interior.Alternatively, the carried agent can be a part of, that is, dispersed ordissolved in the vesicle wall-forming materials.

During Liposome formation, water soluble carrier agents may beencapsulated in the aqueous interior by including them in the hydratingsolution, and lipophilic molecules incorporated into the lipid bilayerby inclusion in the lipid formulation. In the case of certain molecules(e.g., cationic or anionic lipophilic drugs), loading of the drug intopreformed Liposomes may be accomplished, for example, by the methodsdescribed in U.S. Pat. No. 4,946,683, the disclosure of which isincorporated herein by reference. Following drug encapsulation, theLiposomes are processed to remove unencapsulated drug through processessuch as gel chromatography or ultrafiltration. The Liposomes are thentypically sterile filtered to remove any microorganisms which may bepresent in the suspension. Microorganisms may also be removed throughaseptic processing.

If one wishes to encapsulate large hydrophilic molecules with Liposomes,larger unilamellar vesicles can be formed by methods such as thereverse-phase evaporation (REV) or solvent infusion methods. Otherstandard methods for the formation of Liposomes are known in the art,for example, methods for the commercial production of Liposomes includethe homogenization procedure described in U.S. Pat. No. 4,753,788 andthe thin-film evaporation method described in U.S. Pat. No. 4,935,171,which are incorporated herein by reference.

It is to be understood that the therapeutic or diagnostic agent can alsobe associated with the surface of the Lipid Bilayer Vesicle. Forexample, a drug can be attached to a phospholipid or glyceride (aprodrug). The phospholipid or glyceride portion of the prodrug can beincorporated into the lipid bilayer of the Liposome by inclusion in thelipid formulation or loading into preformed Liposomes (see U.S. Pat.Nos. 5,194,654 and 5,223,263, which are incorporated by referenceherein).

It is readily apparent to one skilled in the art that the particularLiposome preparation method will depend on the intended use and the typeof lipids used to form the bilayer membrane.

Lee and Low (1994, JBC, 269: 3198-3204) and DeFrees et al. (1996, JACS,118: 6101-6104) first showed that co-formulation of ligand-PEG-lipidwith lipid components gave liposomes with both inward and outward facingorientations of the PEG-ligand. Passive anchoring was outlined byZalipsky et al. (1997, Bioconj. Chem. 8: 111-118) as a method foranchoring oligopeptide and oligosaccharide ligands exclusively to theexternal surface of liposomes. The central concept presented in theirwork is that ligand-PEG-lipid conjugates can be prepared and thenformulated into pre-formed liposomes via spontaneous incorporation(“anchoring”) of the lipid tail into the existing lipid bilayer. Thelipid group undergoes this insertion in order to reach a lower freeenergy state via the removal of its hydrophobic lipid anchor fromaqueous solution and its subsequent positioning in the hydrophobic lipidbilayer. The key advantage to such a system is that the oligo-lipid isanchored exclusively to the exterior of the lipid bilayer. Thus, nooligo-lipids are wasted by being unavailable for interactions with theirbiological targets by being in an inward-facing orientation.

The efficiency of delivery of a VEGF Nucleic Acid Ligand to cells may beoptimized by using lipid formulations and conditions known to enhancefusion of Liposomes with cellular membranes. For example, certainnegatively charged lipids such as phosphatidylglycerol andphosphatidylserine promote fusion, especially in the presence of otherfusogens (e.g., multivalent cations like Ca²⁺, free fatty acids, viralfusion proteins, short chain PEG, lysolecithin, detergents andsurfactants). Phosphatidylethanolamine may also be included in theLiposome formulation to increase membrane fusion and, concomitantly,enhance cellular delivery. In addition, free fatty acids and derivativesthereof, containing, for example, carboxylate moieties, may be used toprepare pH-sensitive Liposomes which are negatively charged at higher pHand neutral or protonated at lower pH. Such pH-sensitive Liposomes areknown to possess a greater tendency to fuse.

In the preferred embodiment, the VEGF Nucleic Acid Ligands of thepresent invention arc derived from the SELEX methodology. SELEX isdescribed in U.S. patent application Ser. No. 07/536,428, entitledSystematic Evolution of Ligands by Exponential Enrichment, nowabandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10,1991, entitled Nucleic Acid Ligands, now U.S. Pat. No. 5,475,096, U.S.patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitledMethod for Indentifying Nucleic Acid Ligands, now U.S. Pat. No.5,270,163(see also WO 91/19813). These applications, each specificallyincorporated herein by reference, are collectively called the SELEXPatent Applications.

The SELEX process provides a class of products which are Nucleic Acidmolecules, each having a unique sequence, and each of which has theproperty of binding specifically to a desired Target compound ormolecule. Target molecules are preferably proteins, but can also includeamong others carbohydrates, peptidoglycans and a variety of smallmolecules. SELEX methodology can also be used to Target biologicalstructures, such as cell surfaces or viruses, through specificinteraction with a molecule that is an integral part of that biologicalstructure.

In its most basic form, the SELEX process may be defined by thefollowing series of steps:

1) A Candidate Mixture of Nucleic Acids of differing sequence isprepared. The Candidate Mixture generally includes regions of fixedsequences (i.e., each of the members of the Candidate Mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: (a) to assistin the amplification steps described below, (b) to mimic a sequenceknown to bind to the Target, or (c) to enhance the concentration of agiven structural arrangement of the Nucleic Acids in the CandidateMixture. The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

2) The Candidate Mixture is contacted with the selected Target underconditions favorable for binding between the Target and members of theCandidate Mixture. Under these circumstances, the interaction betweenthe Target and the Nucleic Acids of the Candidate Mixture can beconsidered as forming Nucleic Acid-target pairs between the Target andthose Nucleic Acids having the strongest affinity for the Target.

3) The Nucleic Acids with the highest affinity for the target arepartitioned from those Nucleic Acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of Nucleic Acid) corresponding to the highest affinityNucleic Acids exist in the Candidate Mixture, it is generally desirableto set the partitioning criteria so that a significant amount of theNucleic Acids in the Candidate Mixture (approximately 5-50%) areretained during partitioning.

4) Those Nucleic Acids selected during partitioning as having therelatively higher affinity for the target are then amplified to create anew Candidate Mixture that is enriched in Nucleic Acids having arelatively higher affinity for the target.

5) By repeating the partitioning and amplifying steps above, the newlyformed Candidate Mixture contains fewer and fewer unique sequences, andthe average degree of affinity of the Nucleic Acids to the target willgenerally increase. Taken to its extreme, the SELEX process will yield aCandidate Mixture containing one or a small number of unique NucleicAcids representing those Nucleic Acids from the original CandidateMixture having the highest affinity to the target molecule.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, entitled “Method for Selecting Nucleic Acids on theBasis of Structure,” describes the use of SELEX in conjunction with gelelectrophoresis to select Nucleic Acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935,filed Sep. 17, 1993, entitled “Photoselection ofNucleic Acid Ligands,” describes a SELEX based method for selectingNucleic Acid Ligands containing photoreactive groups capable of bindingand/or photocrosslinking to and/or photoinactivating a target molecule.U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993,entitled “High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” now U.S. Pat. No. 5,580,737, describes amethod for identifying highly specific Nucleic Acid Ligands able todiscriminate between closely related molecules, termed Counter-SELEX.U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993,entitled “Systematic Evolution of Ligands by EXponential Enrichment:Solution SELEX,” now U.S. Pat. No. 5,567,588, describes a SELEX-basedmethod which achieves highly efficient partitioning betweenoligonucleotides having high and low affinity for a target molecule.U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992,entitled “Nucleic Acid Ligands to HIV-RT and HIV Rev,” now U.S. Pat. No.5,496,938, describes methods for obtaining improved Nucleic Acid Ligandsafter SELEX has been performed. U.S. patent application Ser. No.08/400,440, filed Mar. 8, 1995, entitled “Systematic Evolution ofLigands by EXponential Enrichment: Chemi-SELEX,” describes methods forcovalently linking a ligand to its target.

The SELEX method encompasses the identification of high-affinity NucleicAcid Ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified Nucleic Acid Ligands containingmodified nucleotides are described in U.S. patent application Ser. No.08/117,991, filed Sep. 8, 1993, entitled “High Affinity Nucleic AcidLigands Containing Modified Nucleotides,” now U.S. Pat. No. 5,660,985,that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S.patent application Ser. No. 08/134,028, supra, describes highly specificNucleic Acid Ligands containing one or more nucleotides modified with2′-amino (2′-NH₂), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S.patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled“Novel Method of Preparation of Known and Novel 2′-Modified Nucleosidesby Intramolecular Nucleophilic Displacement,” describes oligonucleotidescontaining various 2′-modified pyrimidines.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. patent application Ser. No. 08/284,063, filed Aug.2, 1994, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Chimeric SELEX,” now U.S. Pat. No. 5,637,459, and U.S.patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled“Systematic Evolution of Ligands by Exponential Enrichment: BlendedSELEX,” respectively. These applications allow the combination of thebroad array of shapes and other properties, and the efficientamplification and replication properties, of oligonucleotides with thedesirable properties of other molecules.

The SELEX method further encompasses combining selected Nucleic AcidLigands with Lipophilic Compounds or Non-Immunogenic, High MolecularWeight Compounds in a diagnostic or therapeutic Complex as described inU.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled“Nucleic Acid Complexes.” The SELEX method further encompasses combiningselected VEGF Nucleic Acid Ligands with lipophilic compounds, such asdiacyl glycerol or dialkyl glycerol, as described in U.S. patentapplication Ser. No. 08/739,109, filed Oct. 25, 1996, entitled “VascularEndothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes.” VEGFNucleic Acid Ligands that are associated with a High Molecular Weight,Non-Immunogenic Compound, such as Polyethylene glycol, or a LipophilicCompound, such as Glycerolipid, phospholipid, or glycerol amide lipid,in a diagnostic or therapeutic complex are described in U.S. patentapplication Ser. No. 08/897,351, filed Jul. 21, 1997, entitled “VascularEndothelial Growth Factor (VEGF) Nucleic Acid Complexes.” Each of theabove described patent applications which describe modifications of thebasic SELEX procedure are specifically incorporated by reference hereinin their entirety.

SELEX identifies Nucleic Acid Ligands that are able to bind targets withhigh affinity and with outstanding specificity, which represents asingular achievement that is unprecedented in the field of Nucleic Acidsresearch. These characteristics are, of course, the desired propertiesone skilled in the art would seek in a therapeutic or diagnostic ligand.

In order to produce Nucleic Acid Ligands desirable for use as apharmaceutical, it is preferred that the Nucleic Acid Ligand (1) bindsto the target in a manner capable of achieving the desired effect on thetarget; (2) be as small as possible to obtain the desired effect; (3) beas stable as possible; and (4) be a specific ligand to the chosentarget. In most situations, it is preferred that the Nucleic Acid Ligandhas the highest possible affinity to the target. Additionally, NucleicAcid Ligands can have facilitating properties.

In commonly assigned U.S. patent application Ser. No. 07/964,624, filedOct. 21, 1992 ('624), now U.S. Pat. No. 5,496,938, methods are describedfor obtaining improved Nucleic Acid Ligands after SELEX has beenperformed. The '624 application, entitled Nucleic Acid Ligands to HIV-RTand HIV-1 Rev, is specifically incorporated herein by reference.

The SELEX process has been used to identify a group of high affinity RNALigands to VEGF from random 2′-aminopyrimidine RNA libraries and ssDNAligands from random ssDNA libraries (U.S. patent application Ser. No.08/447,169, filed May 19, 1995, entitled High-Affinity OligonucleotideLigands to Vascular Endothelial Growth Factor (VEGF), which is aContinuation-in-Part Application of U.S. patent application Ser. No.08/233,012, filed Apr. 25, 1994, entitled High-Affinity OligonucleotideLigands to Vascular Endothelial Growth Factor (VEGF), both of which areincorporated herein by reference; see also Green et al. (1995) Chemistryand Biology 2:683-695).

In embodiments where the VEGF Nucleic Acid Ligand(s) can serve in atargeting capacity, the VEGF Nucleic Acid Ligands adopt a threedimensional structure that must be retained in order for the VEGFNucleic Acid Ligand to be able to bind its target. In embodiments wherethe Lipid Construct comprises a Complex and the VEGF Nucleic Acid Ligandof the Complex is projecting from the surface of the Lipid Construct,the VEGF Nucleic Acid Ligand must be properly oriented with respect tothe surface of the Lipid Construct so that its target binding capacityis not compromised. This can be accomplished by attaching the VEGFNucleic Acid Ligand at a position that is distant from the bindingportion of the VEGF Nucleic Acid Ligand. The three dimensional structureand proper orientation can also be preserved by use of a Linker orSpacer as described supra.

Any variety of therapeutic or diagnostic agents can be attached to theComplex for targeted delivery by the Complex. In addition, any varietyof therapeutic or diagnostic agents can be attached encapsulated, orincorporated into the Lipid Construct as discussed supra for targeteddelivery by the Lipid Construct.

In embodiments where the Complex is comprised of a Lipophilic Compoundand a VEGF Nucleic Acid Ligand in association with a Liposome, forexample, the VEGF Nucleic Acid Ligand could target tumor cellsexpressing VEGF (e.g., in Kaposi's sarcoma) for delivery of an antitumordrug (e.g., daunorubicin) or imaging agent (e.g., radiolabels). Itshould be noted that cells and tissues surrounding the tumor may alsoexpress VEGF, and targeted delivery of an antitumor drug to these cellswould also be effective.

In an alternative embodiment, the therapeutic or diagnostic agent to bedelivered to the Target cell could be another Nucleic Acid Ligand.

It is further contemplated by this invention that the agent to bedelivered can be incorporated into the Complex in such a way as to beassociated with the outside surface of the Liposome (e.g., a prodrug,receptor antagonist, or radioactive substance for treatment or imaging).As with the VEGF Nucleic Acid Ligand, the agent can be associatedthrough covalent or Non-Covalent Interactions. The Liposome wouldprovide targeted delivery of the agent extracellularly, with theLiposome serving as a Linker.

In another embodiment, a Non-Immunogenic, High Molecular Weight Compound(e.g., PEG) can be attached to the Liposome to provide ImprovedPharmacokinetic Properties for the Complex. VEGF Nucleic Acid Ligandsmay be attached to the Liposome membrane or may be attached to aNon-Immunogenic, High Molecular Weight Compound which in turn isattached to the membrane. In this way, the Complex may be shielded fromblood proteins and thus be made to circulate for extended periods oftime while the VEGF Nucleic Acid Ligand is still sufficiently exposed tomake contact with and bind to its Target.

In another embodiment of the present invention, more than one VEGFNucleic Acid Ligand is attached to the surface of the same Liposome.This provides the a possibility of bringing the same VEGF molecules inclose proximity to each other and can be used to generate specificinteractions between the VEGF molecules.

In an alternative embodiment of the present invention, VEGF Nucleic AcidLigands and a Nucleic Acid Ligand to a different Target can be attachedto the surface of the same Liposome. This provides the possibility ofbringing VEGF in close proximity to a different Target and can be usedto generate specific interactions between VEGF and the other Target. Inaddition to using the Liposome as a way of bringing Targets in closeproximity, agents could be encapsulated in the Liposome to increase theintensity of the interaction.

The Lipid Construct comprising a Complex allows for the possibility ofmultiple binding interactions to VEGF. This, of course, depends on thenumber of VEGF Nucleic Acid Ligands per Complex, and the number ofComplexes per Lipid Construct, and mobility of the VEGF Nucleic AcidLigands and receptors in their respective membranes. Since the effectivebinding constant may increase as the product of the binding constant foreach site, there is a substantial advantage to having multiple bindinginteractions. In other words, by having many VEGF Nucleic Acid Ligandsattached to the Lipid Construct, and therefore creating multivalency,the effective affinity (i.e., the avidity) of the multimeric Complex forits Target may become as good as the product of the binding constant foreach site.

In certain embodiments of the invention, the Complex of the presentinvention is comprised of a VEGF Nucleic Acid Ligand attached to aLipophilic Compound such as a glycerol lipid. In this case, thepharmacokinetic properties of the Complex will be improved relative tothe VEGF Nucleic Acid Ligand alone. As discussed supra, the glycerollipid, phospholipid or glycerol amide lipid may be covalently bound tothe VEGF Nucleic Acid Ligand at numerous positions on the VEGF NucleicAcid Ligand. In embodiments where a glycerol lipid is used, it ispreferred that the VEGF Nucleic Acid Ligand is bonded to the lipidthrough phosphodiester linkages.

In another embodiment of the invention, the Lipid Construct comprises aVEGF Nucleic Acid Ligand or Complex. In this embodiment, theglycerolipid can assist in the incorporation of the VEGF Nucleic AcidLigand into the Liposome due to the propensity for a glycerolipid toassociate with other Lipophilic Compounds. The glycerolipid inassociation with a VEGF Nucleic Acid Ligand can be incorporated into thelipid bilayer of the Liposome by inclusion in the formulation or byloading into preformed Liposomes. The glycerolipid can associate withthe membrane of the Liposome in such a way so as the VEGF Nucleic AcidLigand is projecting into or out of the Liposome. In embodiments wherethe VEGF Nucleic Acid Ligand is projecting out of the Complex, the VEGFNucleic Acid Ligand can serve in a targeting capacity. It is to beunderstood that additional compounds can be associated with the LipidConstruct to further improve the Pharmacokinetic Properties of the LipidConstruct. For example, a PEG may be attached to the exterior-facingpart of the membrane of the Lipid Construct.

In other embodiments, the Complex of the present invention is comprisedof a VEGF Nucleic Acid Ligand covalently linked to a Non-Immunogenic,High Molecular Weight Compound such as Polyalkylene Glycol or PEG. Inthis embodiment, the pharmacokinetic properties of the Complex areimproved relative to the VEGF Nucleic Acid Ligand alone. ThePolyalkylene Glycol or PEG may be covalently bound to a variety ofpositions on the VEGF Nucleic Acid Ligand. In embodiments wherePolyalkylene Glycol or PEG are used, it is preferred that the VEGFNucleic Acid Ligand is bonded through the 5′ hydroxyl group via aphosphodiester linkage.

In certain embodiments, a plurality of Nucleic Acid Ligands can beassociated with a single Non-Immunogenic, High Molecular WeightCompound, such as Polyalkylene Glycol or PEG, or a Lipophilic Compound,such as a glycerolipid. The Nucleic Acid Ligands can all be to VEGF orVEGF and a different Target. In embodiments where there are multipleVEGF Nucleic Acid Ligands, there is an increase in avidity due tomultiple binding interactions with VEGF. In yet further embodiments, aplurality of Polyalkylene Glycol, PEG, glycerol lipid molecules can beattached to each other. In these embodiments, one or more VEGF NucleicAcid Ligands or Nucleic Acid Ligands to VEGF and other Targets can beassociated with each Polyalkylene Glycol, PEG, or glycerol lipid. Thisalso results in an increase in avidity of each Nucleic Acid Ligand toits Target. In embodiments where multiple VEGF Nucleic Acid Ligands areattached to Polyalkylene Glycol, PEG, or glycerol lipid, there is thepossibility of bringing VEGF molecules in close proximity to each otherin order to generate specific interactions between VEGF. Where multipleNucleic Acid Ligands specific for VEGF and different Targets areattached to Polyalkylene Glycol, PEG, or glycerol lipid, there is thepossibility of bringing VEGF and another Target in close proximity toeach other in order to generate specific interactions between the VEGFand the other Target. In addition, in embodiments where there areNucleic Acid Ligands to VEGF or Nucleic Acid Ligands to VEGF anddifferent Targets associated with Polyalkylene Glycol, PEG, or glycerollipid, a drug can also be associated with Polyalkylene Glycol, PEG, orglycerol lipid. Thus the Complex would provide targeted delivery of thedrug, with Polyalkylene Glycol, PEG, or glycerol lipid serving as aLinker.

VEGF Nucleic Acid Ligands selectively bind VEGF. Thus, a Complexcomprising a VEGF Nucleic Acid Ligand and a Non-Immunogenic, HighMolecular Weight Compound or Lipophilic Compound or a Lipid Constructcomprising a VEGF Nucleic Acid Ligand or a Complex are useful aspharmaceuticals or diagnostic agents. The present invention, therefore,includes methods of inhibiting angiogenesis by administration of aComplex comprising VEGF Nucleic Acid Ligand and a Non-Immunogenic, HighMolecular Weight Compound or Lipophilic Compound, a Lipid Constructcomprising VEGF Nucleic Acid Ligand or a Complex comprising a VEGFNucleic Acid Ligand and a Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound. The VEGF Nucleic Acid Ligand-containingComplexes and Lipid Constructs can be used to treat, inhibit, prevent ordiagnose any disease state that involves inappropriate VEGF production,particularly angiogenesis. Angiogenesis rarely occurs in healthy adults,except during the menstrual cycle and wound healing. Angiogenesis is acentral feature, however, of various disease states, including, but notlimited to cancer, diabetic retinopathy, macular degeneration, psoriasisand rheumatoid arthritis. The present invention, thus, also includes,but is not limited to, methods of treating, inhibiting, preventing ordiagnosing diabetic retinopathy, macular degeneration, psoriasis andrheumatoid arthritis. Additionally, VEGF is produced and secreted invarying amounts by virtually all tumor cells. Thus, the presentinvention, includes methods of treating, inhibiting, preventing, ordiagnosing cancer by administration of a Complex comprising a VEGFNucleic Acid Ligand and a Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound, a Lipid Construct comprising a Complex,or a VEGF Nucleic Acid Ligand in association with a Lipid Constructwithout being part of the Complex. It has been shown that in a type ofcancer, Kaposi's sarcoma (KS), cells not only produce abundant amountsof VEGF but also express functional VEGF receptors and therefore useVEGF for autocrine growth. Thus, the present invention includes a methodof inhibiting Kaposi's Sarcoma by administration of a Complex comprisingVEGF Nucleic Acid Ligand and a Non-Immunogenic, High Molecular WeightCompound or a Lipophilic Compound, a Lipid Construct comprising aComplex, or a VEGF Nucleic Acid Ligand in association with a LipidConstruct without being part of a Complex.

In one embodiment of the present invention, the Lipid Constructcomprises a Complex comprised of a VEGF Nucleic Acid Ligand and aLipophilic Compound with an additional diagnostic or therapeutic agentencapsulated in the Lipid Construct or associated with the interior ofthe Lipid Construct. In the preferred embodiment, the Lipid Construct isa Lipid Bilayer Vesicle, and more preferably a Liposome. The therapeuticuse of Liposomes includes the delivery of drugs which are normally toxicin the free form. In the liposomal form, the toxic drug is occluded, andmay be directed away from the tissues sensitive to the drug and targetedto selected areas. Liposomes can also be used therapeutically to releasedrugs over a prolonged period of time, reducing the frequency ofadministration. In addition, liposomes can provide a method for formingaqueous dispersions of hydrophobic or amphiphilic drugs, which arenormally unsuitable for intravenous delivery.

In order for many drugs and imaging agents to have therapeutic ordiagnostic potential, it is necessary for them to be delivered to theproper location in the body, and the liposome can thus be readilyinjected and form the basis for sustained release and drug delivery tospecific cell types, or parts of the body. Several techniques can beemployed to use liposomes to target encapsulated drugs to selected hosttissues, and away from sensitive tissues. These techniques includemanipulating the size of the liposomes, their net surface charge, andtheir route of administration. MLVs, primarily because they arerelatively large, are usually rapidly taken up by thereticuloendothelial system (principally the liver and spleen). UVs, onthe other hand, have been found to exhibit increased circulation times,decreased clearance rates and greater biodistribution relative to MLVs.

Passive delivery of liposomes involves the use of various routes ofadministration, e.g., intravenous, subcutaneous, intramuscular andtopical. Each route produces differences in localization of theliposomes. Two common methods used to direct liposomes actively toselected target areas involve attachment of either antibodies orspecific receptor ligands to the surface of the liposomes. In oneembodiment of the present invention, the VEGF Nucleic Acid Ligand isassociated with the outside surface of the liposome, and serves in atargeting capacity. Additional targeting components, such as antibodiesor specific receptor ligands can be included on the liposome surface, aswould be known to one of skill 25 in the art. In addition, some effortshave been successful in targeting liposomes to tumors without the use ofantibodies, see, for example, U.S. Pat. Nos. 5,019,369, 5,435,989, and4,441,775, and it would be known to one of skill in the art toincorporate these alternative targeting methods.

Therapeutic or diagnostic compositions of a Complex comprising VEGFNucleic Acid Ligand and a Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound, a Lipid Construct comprising a Complexcomprised of a VEGF Nucleic Acid Ligand and a Non-Immunogenic, HighMolecular Weight Compound or Lipophilic Compound, and a VEGF NucleicAcid Ligand in association with a Lipid Construct without being part ofa Complex may be administered parenterally by injection, although othereffective administration forms, such as intraarticular injection,inhalant mists, orally active formulations, transdermal iotophoresis orsuppositories, are also envisioned. One preferred carrier isphysiological saline solution, but it is contemplated that otherpharmaceutically acceptable carriers may also be used. In oneembodiment, it is envisioned that the carrier and the VEGF Nucleic AcidLigand Complex constitute a physiologically-compatible, slow releaseformulation. The primary solvent in such a carrier may be either aqueousor non-aqueous in nature. In addition, the carrier may contain otherpharmacologically-acceptable excipients for modifying or maintaining thepH, osmolarity, viscosity, clarity, color, sterility, stability, rate ofdissolution, or odor of the formulation. Similarly, the carrier maycontain still other pharmacologically-acceptable excipients formodifying or maintaining the stability, rate of dissolution, release, orabsorption of the VEGF Nucleic Acid Ligand. Such excipients are thosesubstances usually and customarily employed to formulate dosages forparental administration in either unit dose or multi-dose form.

Once the therapeutic or diagnostic composition has been formulated, itmay be stored in sterile vials as a solution, suspension, gel, emulsion,solid, or dehydrated or lyophilized powder. Such formulations may bestored either in ready to use form or requiring reconstitutionimmediately prior to administration. The manner of administeringformulations containing VEGF Nucleic Acid Ligand for systemic deliverymay be via subcutaneous, intramuscular, intravenous, intranasal orvaginal or rectal suppository.

The advantages of the Complexes and Lipid Constructs of the inventioninclude: i) improving the plasma pharmacokinetics of the Nucleic AcidLigand; ii) presenting Nucleic Acid Ligands in a multivalent array withthe aim of increasing the avidity of interaction with their targets;iii) combining two or more presenting Nucleic Acid Ligands withdifferent specificities in the same liposome particle; iv) enhancing thedelivery of presenting Nucleic Acid Ligands to tumors by takingadvantage of the intrinsic tumor targeting properties of liposomes; andv) using the high affinity and specificity of presenting Nucleic AcidLigands, which is comparable to that of antibodies, to guide liposomalcontents to specific targets. Presenting Nucleic Acid Ligands are wellsuited for the kinds of preparations described here since, unlike mostproteins, the denaturation of presenting Nucleic Acid Ligands by heat,various molecular denaturants and organic solvents is readilyreversible.

The following examples are provided to explain and illustrate thepresent invention and are not to be taken as limiting of the invention.The structures of the Nucleic Acid Ligands described in the examplesbelow are shown in FIG. 1. Example 1 describes the conjugation ofNucleic Acid Ligands with lipid reagents. The ability of adialkylglycerol derivative of the VEGF Nucleic Acid Ligand (NX278),either as a free ligand or incorporated in the bilayer of liposomes(NX278-L), to inhibit the activity of VEGF in vitro and in vivo isdescribed in Example 2. Example 3 describes the experimental proceduresfor generating 2′-F pyrimidine modified RNA ligands to VEGF. Example4describes the 2′-F pyrimidine-modified RNA ligands to VEGF. Example 5describes the synthesis of glycerolipid, phospholipid, and glycerolamide lipid, and PEG-modified VEGF Nucleic Acid Ligands. Example 6describes the pharmacokinetic properties of phospholipid (PL) and PEGmodified VEGF Nucleic Acid Ligands. Example 7 describes preparations ofNX3 1838 PL-Liposome Complex. Examples 8-10 describe the in vivoefficacy of VEGF Nucleic Acid Ligand Complexes. Example 11 describes theintravitreal pharmacokinetics of NX31838-40K PEG in rabbits.

Example 1 Synthesis of a dialkyl glycerol(1,2-di-O-octadecyl-sn-glycerol)-modified VEGF Nucleic Acid Ligand

In this example, conjugation of Nucleic Acid Ligands with lipid reagentsis described. Synthesis of (1,2-di-O-octadecyl-sn-glycerol)—modifiedVEGF Nucleic Acid Ligand is shown below.

Tetraethylene glycol monotosylate (2a)

Tetraethylene glycol (200 mL, 1.15 mol) was dissolved in 500 mL ofpyridine and cooled to 0° C. and treated with 22.0 g (0.115 mol) ofp-toluenesulfonyl chloride. When solution was complete, the reactionmixture was stored in the refrigerator overnight, and then concentratedin vacuo. The residue was dissolved in 800 mL of EtOAc and extractedwith 3×600 mL of H₂O. The H₂O fractions were back-extracted with EtOAc,and the combined EtOAc fractions were extracted with saturated aqueousNa₂HPO₄. The organic phase was dried over MgSO₄ and concentrated to acolorless oil. The oil was purified by flash chromatography using 800 mLof silica gel and eluting with hexane, 25% EtOAc-50% EtOAc in hexane,then EtOAc, then 10% MeOH-20% MeOH in EtOAc to afford 23.7 g (60%) ofpure product and 11% of product containing a minor impurity. 2a: ¹H NMR(300 MHz, CDCl₃) d 7.77 (d, J=8.1 Hz, 2H), 7.32 (d, J=8.1 Hz, 2H), 4.13(t, J=4.8 Hz, 2H), 3.68-3.53 (m, 14H), 2.58 (t, J=5.6 Hz, 1H), 2.42 (s,3H); ¹³C NMR (75 MHz, CDCl₃) d 168.2, 158.3, 144.8, 135.9, 133.8, 132.0,129.9, 128.0, 127.7, 126.6, 123.1, 113.0, 85.9, 73.0, 70.6, 70.4, 70.0,69.7, 67.8, 64.4, 55.1, 37.1; Low resolution MS m/e calculated forC₁₅H₂₄O₈S (M+1): 349.1.

Tetraethylene glycol monophthalimide (3a)

To a stirred solution of 31.96 g (0.092 mol) of 2a in 400 mL ofanhydrous DMF was added 14.2 g (1.05 equiv.) of phthalimide and 14.4 mL(1.05 equiv.) of 1,8-diazabicyclo[5.4.0]undec-7-ene. The solution washeated at 70° C. for 18 h then concentrated in vacuo. The crude yellowoil was purified by flash chromatography using 1600 mL of silica gel andeluting with 25% EtOAc-50% EtOAc-75% EtOAc in hexane, then EtOAc, then10% MeOH-20% MeOH in EtOAc to afford 23.8 g (80%) of 3a as an oil. Uponstanding, 3a became a waxy white solid. ¹H NMR (300 MHz, CDCl₃) d7.84-7.78 (m, 2H), 7.70-7.66 (m, 2H), 3.86 (t, J=5.6 Hz, 2H) 3.70 (t,J=5.6 Hz, 2H), 3.64-3.51 (m, 12H), 2.67 (bs, 1H); ¹³C NMR (75 MHz,CDCl₃) d 168.2, 133.8, 132.0, 123.1, 72.4, 70.5, 70.4, 70.2, 70.0, 67.8,61.6, 37.2.

Synthesis of compound 4a

A solution of 15 g (0.0464 mol) of 3a in 150 mL of THF and 15 ml, of DMFwas cooled to 0° C. under Ar. Allyl bromide (6.0 mL, 1.5 equiv.) wasadded to the solution, followed by addition of 1.76 g (1.5 equiv.) ofNaH as a solid. The opaque yellow suspension was stirred at 0° C. for 30minutes and then at room temperature for 18 hr. MeOH (50-100 mL) wasadded and concentrated then mixture was concentrated in vacuo. The crudematerial was purified by flash chromatography using 1500 mL of silicagel and eluting with 25% EtOAc-50% EtOAc-75% EtOAc in hexane, thenEtOAc, then 10% MeOH in EtOAc to afford 11.05 g (65%) of 4a as a yellowoil. ¹H NMR (300 MHz, CDCl₃) d 7.84-7.80 (m, 2H), 7.72-7.67 (m, 2H),5.94-5.84 (m, 1H), 5.28-5.14 (m, 2H). 3.99 (d, J=5.61 Hz, 2H), 3.88 (t,J=5.85 Hz, 2H), 3.72 (t, J=5.76 Hz, 2H), 3.64-3.54 (m, 13H); ¹³C NMR (75MHz, CDCl₃) d 168.0, 134.6, 133.7, 131.9, 123.0, 116.9, 72.0, 70.4,69.9, 69.2, 67.7, 37.0.

1-Dimethoxytrityl-3-(phthalimidotetraethyleneglycolyl)-sn-glycerol (9)

According to Scheme 1, compound 9 was synthesized as follows: To astirred solution of 4a (10.13 g, 0.0279 mol) in 100 mL of acetone and 1mL of H₂O was added 3.98 g (1.22 equiv.) of N-methylmorpholineN-oxide.To this suspension was added 1.75 mL (0.005 equiv.) of Osmium tetroxideas a 2.5% solution in iPrOH. After addition of the OsO₄ solution, thereaction mixture became clear yellow. After TLC analysis indicatedcomplete conversion of 4a (ca 16 h), the reaction mixture was treatedwith 1.5 g of sodium hydrosulfite and 5.0 g of florisil and stirred 30minutes. The suspension was filtered through florisil, the filtrate wasconcentrated to an oil. This crude product was combined with anotherbatch prepared in the same manner from 1.0 g of 4a. Two 100 mL portionsof pyridine were co-evaporated from the combined lots and the residuewas dissolved in 300 mL pyridine. The solution was cooled to 0° C. and10.89 g (1.05 equiv.) of 4,4′-dimethoxytrityl chloride was added. Adrying tube was inserted in the flask and the reaction mixture wasstirred at room temperature 16 h. The solution was treated with 20 mL ofMeOH and concentrated in vacuo, keeping the temperature of the waterbath below 40° C. The crude oil was purified by flash chromatographyusing 1100 mL of silica gel (wet-packed onto column using 3%triethylamine in hexane) and eluting with 10-100% EtOAc in hexane (allcontaining 3% triethylamine)to give 21.3 g (89% after two steps) of 9 asa yellow oil. ¹H NMR (300 MHz, CDCl₃) d 7.80-7.77 (m, 2H), 7.66-7.64 (m,2H), 7.39-7.22 (m, 9H), 7.20-6.76 (m, 4H), 3.97 (bs, 1H), 3.84 (t,J=5.97 Hz, 2H), 3.74 (s, 6H), 3.68 (t, J=5.7 Hz, 2H), 3.60-3.49 (m,14H), 3.13-2.76(m, 2H), 2.00 (bs, 1H); ¹³C NMR (75 MHz, CDCl3) d 168.2,158.3, 144.8, 135.9, 133.8, 132.0, 129.9, 128.0, 127.7, 126.6, 123.1,113.0, 85.9, 73.0, 70.6, 70.4, 70.0, 69.7, 67.8, 64.4, 55.1, 37.1; Lowresolution MS m/e calculated for C₄₀H₄₅O₁₀N (M+NH₄+): 717.5.

1-Dimethoxytrityl-3-(aminotetraethyleneglycolyl)-sn-glycerol (10)

According to Scheme 1, compound 10 was synthesized as follows: Compound9 (5.2 g, 7.2 mmol) was taken up in 50 mL of 40% methylamine in H₂O and10 mL of methanol was added to solubilize the starting material. Thereaction mixture was heated at 50° C. for 5 hr, and then wasconcentrated in vacuo and coevaporated with toluene. The crude materialwas purified by flash chromatography on 200 mL of silica gel, elutingwith 15% methanolic ammonia in dichloromethane. Collected 3.94 g (96%)of 10 as a pale yellow oil. ¹H NMR (300 MHz, CDCl₃) d 7.46-7.21 (m, 9H,DMT), 6.81 (d, 4H, DMT), 4.00 (m, 1H), 3.80 (s, 6H), 3.70-3.49(overlapping m, 18H), 3.20 (dd, J=9.24, 5.49 Hz, 1H), 3.12 (dd, J=9.21,6.0 Hz, 1H), 2.84-2.80 (m, 3H); ¹³C NMR (75 MHz, CDCl₃) d 158.30,144.82, 136.01, 129.95, 128.04, 127.66, 126.61, 112.95, 85.85, 73.46,72.85, 70.55, 70.45, 69.99, 69.51, 64.43, 55.10, 41.40; Low resolutionMS m/e calculated for C₃₂H₄₄O₈N (M+1+): 570.353, found 570.4.

Chloroformate 19: To a stirred solution of 3 g (5.03 mmol) of1,2di-O-octadecyl-sn-glycerol 18 in 60 mL of toluene was added 20 mL ofa 1.93 M solution of phosgene. Additional phosgene solution (2×10 mL;15.4 equiv phosgene total) was added until no further alcohol startingmaterial remained (by ¹H NMR analysis of concentrated aliquots). Theexcess phosgene and HCl was removed by aspirator and the reactionmixture was concentrated in vacuo to afford 3.3 g (98%) of the desiredchloroformate 19 as a white powder. ¹H NMR (300 MHz, CDCl₃) d 4.45 (dd,J=11.22, 3.69 Hz, 1H), 4.34 (dd, J=11.22, 6.15 Hz, 1H), 3.65 (m, 1H),3.56-3.40 (m, 6H), 1.53 (m, 4H), 1.24 (m, 62H), 0.87 (t, J=6.36 Hz, 6H);¹³C NMR (75 MHz, CDCl₃) d 75.90, 71.91, 71.35, 70.93, 69.36, 31.99,29.96-29.44 (overlapping signals from hydrocarbon chains), 26.13, 26.04,22.76, 14.18.

Conjugate 20: To a stirred solution of 2.25 g (3.95 mmol) of 10 in 60 mLof pyridine was added 2.6 g of the distearyl glycerol chlorofornate 18.¹H NMR analysis of a concentrated aliquot after 2 h revealed noremaining chloroformate and the mixture was concentrated in vacuo. Thecrude residue was combined with material similarly prepared from 0.5 g(0.88 mmol) of 10 and 0.58 g of the chloroformate and the combined lotspurified by flash silica gel chromatography on a column of 100 mL ofsilica gel (packed in hexanes containing 2% triethylamine) eluting with200 mL hexanes, then 250 mL each of 10-20 and 30% EtOAc in hexanes, 500mL 40% EtOAc in hexanes, then 250 mL each of 50-60-70 and 80% EtOAc inhexanes, and finally with 250 mL of EtOAc. The product containingfractions were concentrated to afford 3.3 g (57%) of the conjugate 20.

Phosphoramidite21: To a stirred solution of 3.8 g (3.26 mmol) of theconjugate in 25 mL of CH₂Cl₂ was added 1.14 mL (6.52 mmol) ofdiisopropylethylaminethen 1.09 mL (4.88 mmol) of 2-cyanoethylN,N-diisopropylchloro-phosphoramidite. After 2 hours, the mixture wasdiluted with CH₂Cl₂ and washed with saturated NaHCO₃ solution, driedover Na₂SO₄, and concentrated, The crude residue was purified by flashsilica gel chromatography on a column of 125 mL of silica gel (packed inhexanes containing 2% triethylamine) eluting with 100 mL hexanes, then250 mL each of 10 and 20% EtOAc in hexanes, 500 mL 30% EtOAc in hexanes,then 250 mL of 50% EtOAc in hexanes. The product containing fractionswere concentrated to afford 4.2 g (95%) of the phosphoramidite21. ³¹PNMR (CDCl₃) d 151.52, 151.08.

The VEGF Nucleic Acid Ligand-1,2-di-O-octadecyl-sn-glycerolconjugate

The 1,2-di-O-octadecyl-sn-glycerolgroup was conjugated to VEGF NucleicAcid Ligand NX213 (See FIG. 1A) using phosphoramidite 21 (Scheme 2). Theresulting conjugate was named NX278 (See FIG. 1B). NX278 (SEQ ID No: 2)was purified by reverse phase HPLC and its composition was confirmed byelectrospray mass spectroscopy (m/z observed=11703″±4, m/zcalculated=11720). Phosphorothioate internucleoside linkages were usedat 8 positions in NX278 (at the 3′ and 5′ ends) and the difference of0.16 mass units between the expected and observed masses is probably dueto incomplete oxidation by the sulfurizing agent resulting, on average,in one less phosphorothioate linkage per molecule than expected.

Example 2

In vitro and in vivo efficacy of Nucleic Acid Ligand-Liposome Complex.Dialkylglycerol(DAG)-modified VEGF Nucleic Acid Ligand (NX278) embeddedin Liposome bilayer.

NX278-Liposome Complex was prepared by incubating NX-278 (1 mg) (FIG.1B; SEQ ID NO: 2) with a spray-dried mixture of DSPC:cholesterol(50mg/ml; 2:1, Mol:Mol) in 25 mM phosphate (pH 7.4) buffer containing 9%sucrose and sonicated for 15-30 min at approximately 60 degrees C. usinga probe-type sonicator until opalescent solution was obtained. Thecontrol Nucleic Acid Ligand-Liposome Complex containing a sequencescrambled analog of ligand NX-278 (scNX278) (FIG. 1C; SEQ ID NO:3) wasprepared in the same manner. In a typical preparation, liposomes with amean diameter of 50 nm and a distribution width at half height of 20 nmwere obtained. The size of Liposome particles was determined in aparticle analyzer (Leeds & Northrup Model Microtrack UPA 150, Horsham,Pa.). Liposomes of comparable size distribution were obtained with thesame lipid composition but without the lipid-conjugated Nucleic AcidLigand. A 50 nm liposome is expected to contain an average of 40 NucleicAcid Ligands, displayed on both sides of the bilayer. The calculationwas made as follows. Assuming a surface area of 19 Å for cholesterol and60 Å for distearylphoshatidylcholine in the liposome, a number of lipidmolecules per liposome of 3.13×10⁴ was obtained, for a sphericalliposome with 50 nm outer diameter and membrane thickness of 20 Å. Fromthe composition of the liposome (2:1 mol:moldistearyphosphatidylcholine(MW=790.2):cholesterol(MW=386.7)), assuminghomogeneous distribution of lipids, molecular mass of 2.1×10⁷ for theliposome was calculated.

To determine the partitioning of the Nucleic Acid Ligands between theinside and outside surfaces of liposomes, the accessibility of NX278 inthe liposomal formulation to T₁ ribonuclease was examined. With tworiboguanosines in the sequence (Green et al. (1995) Chemistry andBiology 2:683-695), NX278 is efficiently cleaved by ribonuclease T₁.Simple incubation of NX278 with preformed liposomes does not protect theNucleic Acid Ligand from ribonuclease T₁. However, when NX278 isincorporated in liposomes by sonication (NX278-Liposome), about ⅓ isprotected from the nuclease. The addition of 0.1% triton X-100 toNX278-Liposome, which disrupts the liposomes without affecting theactivity of the nuclease, exposes the previously protected Nucleic AcidLigand to digestion. These results are consistent with the notion thatthe Nucleic Acid Ligand is distributed on both sides of the bilayer.

Binding Affinities of NX213, NX278, and NX278-Liposome for VEGF

The binding affinities of NX213, NX278 and NX278-Liposome for VEGF wereexamined using a competition electrophoretic mobility shift method (FIG.2). The binding affinity of NX278 for VEGF was comparable to that ofNX213. The apparent binding affinity of NX278-Liposome was 3-fold lowercompared with NX278. A part of the observed affinity reduction ispotentially due to the confinement of a fraction of the Nucleic AcidLigand to the liposome interior. As expected, the sequence scrambledanalogs bind to VEGF with substantially lower affinities (FIG. 2).

Plasma Pharmacokinetic Properties of NX213, NX278, and NX278-Liposome

The concentrations of NX213, NX278 and NX278-Liposome in the plasma ofSprague Dawley rats as a function of time are shown in FIG. 15, and theparameters from compartmental analysis are summarized in Table 1. Themajority of NX213 is cleared rapidly in the alpha phase with a t_(1/2)of 7 minutes and an overall clearance rate of 6.8 ml/kg/min. Conjugationof a phospholipid group to the Nucleic Acid Ligand results in highlybiphasic clearance from the blood with increased β(t_(1/2)) and somewhatslower overall rate of clearance (4.95 ml/kg/min) relative to NX213.Incorporation of NX278 into a liposome shows a substantial additionaldecrease in clearance of the Nucleic Acid Ligand from plasma (1.88ml/kg/min).

The Effect of NX278 on HUVEC Proliferation and Angiogenesis

The effects of NX278-liposome, scNX278-liposome and NX213 on theproliferation of human umbilical vein endothelial cells (HUVEC) wasexamined. HUVECs were grown in the presence of VEGF (10 ng/ml) inIMDM:Ham's F12 (1:1) medium containing 10% fetal calf serum (FCS) andheparin (45 μg/ml). Cells were plated in 24-well gelatin-coated platesat a density of 20,000 cells per well on day zero and treated with theabove ligands at concentrations between 0.1 nM to 1 μM on days 1, 2, and3 (replacing the media along with the ligands. NX278-Liposome inhibitedthe proliferation of HUVECs with an IC50 of ≈300 nM (the concentrationrefers to the Nucleic Acid Ligand component); scNX278-Liposome and NX213were significantly less effective (IC50>1 μg).

VEGF induces angiogenesis in chicken allantoic membrane (CAM) assays,and this assay can be utilized to study compounds that inhibitangiogenesis. The assay is done by placing filter discs soaked in VEGFon the CAM and the development of new blood vessels can be quantitiated.NX278-Liposome effectively blocked VEGF induced angiogenesis (data notshown), while NX213, NX278, and scNX278-Liposome had no effect. Togetherthese studies demonstrate that NX278 is a specific inhibitor of VEGFinduced endothelial cell proliferation in vitro and new vessel formationin vivo.

Effect of NX278 on VEGF Induced Capillary Permeability

VEGF is the only known angiogenic factor that transiently enhancescapillary permeability. The ability of NX278-Liposome to inhibit thevascular permeability activity of VEGF in vivo was examined. Thevascular permeability assay (also known as the Miles assay (Miles, A. A.and Miles, E. M. (1952) J. Physiol. (London) 118:228) was performed inguinea pigs essentially as described (Senger, R. S. et al., (I 1983)Science 219:983). NX278-Liposome, NX278, and NX213 at the concentrationof 1 μM were injected intradermally with VEGF (20 nM) in guinea pigspreinjected with Evans blue dye. In response to VEGF, an increase invascular permeability causes extravasation of albumin-bound Evans bluedye resulting in a blue spot at the site of injection. Because therecovery of the dye by organic solvent extraction is generally verypoor, a quantitation method has been developed that measures theabsorption of light through the skin. NX213, NX278, NX278-Liposome andneutralizing monoclonal antibody to VEGF all significantly inhibitedVEGF-induced permeability as shown in FIG. 3. Among the Nucleic AcidLigands, NX278-Liposome appeared to be the most potent antagonist.Sequence scrambled analogs of these compounds were not inhibitory. Thedifferences were dramatic and noticeable to the naked eye.

NX278-L Inhibits Kaposi's Sarcoma Cell Lines in vitro

Inhibitors of VEGF have a potential utility in a variety of diseases,including malignancies where tumor progression and metastasis aredependent on new vessel formation. While most tumor types are known toproduce VEGF, previously none has been shown to express functional VEGFreceptors. It has been shown recently that Kaposi's Sarcoma (KS) cellsnot only produce abundant amounts of VEGF but also express functionalVEGF receptors and therefore use VEGF for autocrine growth. KS celllines thus provide a unique opportunity to examine the ability of NX278to interrupt the autocrine VEGF growth activity.

The effects of NX278-Liposome, scNX278-Liposome and NX213 on theproliferation of KS cells was examined. KS cell line KSY-1 was plated in24-well gelatin coated plates at a density of 7,500-10,000 cells perwell on day zero in medium containing RPMI 1640 supplemented with 2%FCS, L-glutamine, penicillin and streptomycin. Nucleic Acid Ligands wereadded at concentrations between 0.1 nM to 1 μM in fresh medium on day 1,2, and 3 and the cell count was performed on day 4. NX278-Liposomeinhibited the proliferation of KS cells with an IC50 of 100 nM; at 1 MNX278-Liposome, the growth of these cells was completely inhibited.scNX278-Liposome and NX213 exhibited IC50 values of >1 μM (FIG. 4).

NX278-Liposome Inhibits KS Cell Growth in vivo

Because VEGF is a growth factor for KS cells, the effect of VEGFanatagonists on KS tumors in vivo is likely to be two-fold: inhibitionof paracrine growth effect of VEGF on tumor associated endothelial cellsand inhibition of autocrine growth effect on tumor cells. KS tumors maythus be particularly sensitive to VEGF antagonists. To test the activityof the Nucleic Acid Ligands in vivo, tumor trocars (3 mm³) wereimplanted in athymic mice on day one and treated for five consecutivedays beginning on day two with 50, 100 or 150 μg/day/mouse. The rate oftumor growth was measured for a period of two weeks. NX278-Liposomeinhibited the tumor growth in a dose dependent manner with very littleinhibition of tumor growth at the lowest dose level of 50 μg/day/mousedose (FIG. 5A), and marked inhibition of tumor growth at both 100 and150 μg/day/mouse dose levels (FIG. 5B, 150 μg/day/mouse shown). Emptyliposomes (FIGS. 5A, B), scNX278-Liposome as well as NX213 and NX278were ineffective at all doses examined. In addition, NX278-Liposomeblocked the VEGF-induced fluid leakage from blood vessels.

Example 3 Experimental Procedures for 2′-Fluoro Pyrimidine-Modified RNALigands to VEGF

This example provides general procedures followed and incorporated inExample 4 for the evolution of 2′-Fluoro-modified Nucleic Acid Ligandsto VEGF.

Materials

Recombinant human VEGF₁₆₅ purified from the insect cell-line Sf21 waspurchased from R & D Systems as a carrier-free lyophilized powder. Theprotein was resuspended in phosphate-buffered saline to a concentrationof 10 μM and stored at −20° C. in small aliquots until use. Aliquotswere stored at 4° C. for up to 4 weeks after thawing. Sf21-expressedmouse VEGF₁₆₄, and E. coli-expressed human VEGF₁₂₁, VEGF/PIGFheterodimer, and PIGF were also purchased from R & D Systems ascarrier-free, lyophilized preparations.

Oligonucleotides were purchased from Operon Technologies, Inc. or weresynthesized using an Applied Biosystems Model 394 oligonucleotidesynthesizer according to optimized protocols. 2′-F-and2′-OMe-ribonucleotide phosphoramidites were prepared by JBL Scientific,Inc. (San Luis Obispo, Calif.). 2′-F-pyrimidine NTPs were also purchasedfrom JBL. 2′-OH-purine NTPs and dNTPs were from Pharmacia Biotech,Piscataway, N.J.

T. aquaticus thermostable DNA polymerase (Taq polymerase) was purchasedfrom Perkin Elmer-Cetus, (Foster City, Calif.); AMV reversetranscriptase (AMV RT) was from Life Sciences, Inc.; Klenow DNApolymerase was from New England Biolabs, Beverly, Mass. T7 RNApolymerase was from Enzyco, Inc. (Denver, Colo.). Sequenase DNApolymerase is produced by United States Biochemical Corp. (Cleveland,Ohio.).

α-[³²P]-ATP and γ-[³²P]-ATP were obtained from New England Nuclear(Boston, Mass.).

The SELEX Protocol

The SELEX procedure has been described in detail in the SELEX PatentApplications. Chemically synthesized DNA oligonucleotide libraries(“30N7” and “40N7”) were prepared with randomized regions of 30 or 40nucleotides flanked by common 5′ and 3′ fixed sequences(5′-TAATACGACTCACTATAGGGAGGACGATGCGG(30 or 40 N) CAGACGACTCGCCCGA-3′;SEQ ID NO: 133 and 134). Italicized nucleotides at the 5′ end of eachtemplate correspond to the T7 RNA polymerase promoter sequence.Oligonucleotide primers were also synthesized for use in templatepreparation and amplification, and reverse transcription:5′-TCGGGCGAGTCGTCTG-3′ (“3N7”; SEQ ID NO: 135) and5′-TAATACGACTCACTATAGGGAGGACGATGCGG-3′ (“5N7” SEQ ID NO:136).Double-stranded DNA templates were prepared by annealing primer 3N7 tothe 30N7 or 40N7 libraries and extending the primer using Klenow DNApolymerase or AMV RT. The higher temperature of incubation used for AMVRT (45° C. rather than 37° C.) may better promote complete extensionthrough highly structured template oligonucleotides. The libraries weretranscribed using T7 RNA polymerase in the presence of 1 mM each2′-OH-ATP and GTP, 3 mM each 2′-F-CTP and UTP, and 50 μCi α-³²P-ATP.RNAs were purified from denaturing polyacrylamide gels by excising thegel slice containing the RNA, crushing it, and soaking for an extendedtime in 2 mM EDTA.

The SELEX process of affinity selection followed by selected poolamplification has been described in detail (See the SELEX PatentApplications). In brief, one round of selection and amplification wasperformed as follows: VEGF was mixed with a 5- or 10-fold excess of RNAin phosphate-buffered saline with 1 mM MgCl₂ (PBSM) (30N7 and 40N7libraries) or in Tris-buffered saline, 1 mM MgCl₂, 1 mM CaCl₂ (TBSMC)(30N7 library only), and the mixture was serially diluted by three.After incubation at 37° C. for 15 minutes, the mixtures were passedthrough 0.45μ Type HA filters (Millipore) to collect complexes of VEGFwith RNA. RNAs were eluted from selected filters by incubation in 2:1phenol, pH 7:7 M urea. After precipitation from the aqueous phase, RNAswere annealed to primer 3N7 and reverse transcribed using AMV RT. Theresultant cDNAs were amplified with 15 cycles of the polymerase chainreaction (PCR) using the 3N7 and 5N7 primers and Taq DNA polymerase.Transcription of the PCR product yielded a new library enriched forsequences with affinity for VEGF. At round 4, a substantial backgroundfilter-binding signal in the absence of VEGF had emerged in all threeselected RNA pools. To deplete the pools of filter-binding RNAs, rounds5 and 6 were performed with an alternative scheme for partitioningVEGF-bound RNAs from unbound molecules: after incubation of the RNA poolwith the growth factor, each mixture was applied to an 8%polyacrylamide, non-denaturing gel and electrophoresed at 10 W for 45-60minutes at 4° C. VEGF/RNA complexes migrated above the unbound RNA inthis system and were visualized by exposure of X-ray film to the gel.For these rounds, selected RNAs were purified by the crush and soakmethod, as described above. After twelve rounds of selection andamplification, individual molecules in the selected pools were clonedusing the pCR-Script Direct Cloning kit from Stratagene (La Jolla,Calif.). Plasmids were purified using the alkaline lysis method(PERFECTprep Plasmid DNA kit, 5 Prime→3 Prime, Boulder, Colo.) andsequences of the cloned regions were obtained using the Dye TerminatorCycle Sequencing kit available from Perkin Elmer (Foster City, Calfi.).Fluorescent sequencing ladders were read at the National Jewish Center,laboratory of Brian Kotzin, Denver, Colo. Sequences were grouped intofamilies and aligned by eye.

Measurement of Binding Affinities

Nucleic Acid Ligands radiolabeled during transcription by incorporationof α-[³²P]-labeled NTPs, or after synthesis using γ-[³²P]-ATP and T4polynucleotide kinase, were incubated in low concentration (between 20and 71 pM) with varying concentrations of VEGF or other growth factorsat 37° C. for 15 minutes. Incubations were in TBS, PBS, orHEPES-buffered saline (HBS), pH 7.4, with or without the addition ofsupplemental divalent cations. Samples were passed through prewashed0.45μ Type HA filters (Millipore) followed by a 5-10 ml wash withbinding buffer. Filters were immersed in scintillant and counted toquantitate the amount of protein-bound RNA retained by each filter. Theequilibrium dissociation constant (K_(D)) of Nucleic Acid Ligand bindingto a specific protein was calculated from the data points as describedin Green et al. (1996) Biochem. 35:14413-14424.

Affinity Selection of Nucleic Acid Ligand Fragments

Ten pmol internally-radiolabeled transcripts of high affinity VEGFNucleic Acid Ligands were partially digested with S7 nuclease togenerate a mixture of radiolabeled fragments. One-tenth of thefragmented RNA was incubated with 10 pM VEGF in 45 ml binding buffer,prior to filtration through nitrocellulose. Selected fragments recoveredfrom the filter were run out on a high resolution denaturingpolyacrylamide gel next to a lane loaded with the unselected fragmentpool. The smallest selected bands were individually purified from thegel and further labeled at their 5′ ends with polynucleotide kinase toincrease their specific activity. One-half of the sample was annealed toa cDNA of the original transcript and extended to the end of thetemplate using Sequenase DNA polymerase. Comparison of the migration ofthe purified fragment and its extension product to a standard sequencingladder was used to determine the probable size and position of theselected fragment within the original transcript. Syntheticoligonucleotides corresponding in sequence to the affinity selectedfragments were prepared to verify that the truncated Nucleic Acid Ligandretained affinity for VEGF.

2′-OMe-substitution

The 2′-OMe substitution experiments were performed essentially asdescribed in Green et al. (1995) Chem. Biol. 2:683-695. Three or fourlibraries were prepared for each of three truncated ligands (t22, t2,t44) in which five or six 2′-OH-purine positions were partially2′-OMe-substituted. Each purine position was partially 2′-OMe-modifiedin only one of the libraries. Each 5′-radiolabeled library was incubatedwith VEGF, and substituted oligonucleotides bound by the protein werecollected on nitrocellulose filters. The selected pool and the startingunselected library were partially hydrolyzed by alkali and the productswere displayed on a high resolution polyacrylamide gel. A “bandintensity ratio” was determined for each purine position by dividing thephosphorimage signal obtained from hydrolysis at that position in theselected pool by the signal obtained for the same position in theunselected library. Band intensity ratios that fall well above the rangefor a particular position are indicative of a bias for 2′-OH (against2′-OMe) in the affinity selected pool.

Binding Rate Constants

A small amount (typically less than 1 pmol) of 5′-radiolabeled NucleicAcid Ligands were incubated with 1 nM VEGF at 37° C. in 1 ml bufferedsaline supplemented with divalent cations. At time “zero,” 50 μl werefiltered through nitrocellulose to determine the fraction of RNA boundto protein, then an excess (100 or 500 nM in different experiments) ofunlabeled Nucleic Acid Ligand was added and 50 μl aliquots were filteredat time points thereafter. Filters were counted in scintillant todetermine the amount of radiolabeled RNA still bound to VEGF at eachtime point. The data, plotted as fraction of RNA bound (f) vs time, wasfitted to an equation for exponential decay:

f(t)=f ₀ e ^(−kt) +b,

where f₀ is the fraction of RNA bound at time zero, k is thedissociation rate constant (k_(d)) and b is the residual binding ofradiolabeled RNA to the filter at the end of the experiment(effectively, in the absence of protein). Association rate constants(k_(a)s) were calculated from the measured k_(d) and K_(D) valuesaccording to the equation:

k _(a) =k _(d) /K _(D)

Example 4 2′-Fluoro-modified RNA Ligands to VEGF Selection of Ligands

Ligands to VEGF were isolated in three separate SELEX experiments fromlibraries of 2′-F-pyrimidine-modified RNAs containing 30 or 40 randomnucleotides. Selections were performed in PBS supplemented with 1 mMMgCl₂ (30N and 40N libraries) or in Tris-buffered saline with 1 mM MgCl₂and 1 mM CaCl₂ (30N library only). Approximately 10¹⁴ unique sequenceswere included in the first selection cycle of each experiment. After tencycles, the affinity between VEGF and each RNA pool had improvedapproximately 1000-fold relative to the starting pools. As no furtherimprovement in binding affinity was observed after two additionalcycles, individual members of the twelfth round pools were cloned andsequences were determined for about 50 isolates from each selection.

Oligonucleotide ligands to VEGF₁₆₅ were isolated in three separate SELEXexperiments. Individual clones were isolated and sequenced and thesequences grouped into families based on shared primary structuralmotifs (Table 2). The name of each ligand indicates the target (V=VEGF),the selection buffer (P=PBS; T=TBS), the length of the randomized regionin the library (30 or 40 nucleotides) and the clone number (followingthe decimal). The frequency with which a sequence appeared among theclones analyzed is indicated in parentheses; sequences that differed byonly one nucleotide were attributed to PCR mutagenesis of a commonprecursor and were grouped together with the variable base indicated inthe sequence by the appropriate symbol (Y=U or C). The fixed sequencescommon to all ligands are shown in lower case letters at the top. Forindividual clones the sequence of the variable region is shown in uppercase. For some ligands, fixed region sequences in lower case areappended to the variable region sequence where they contribute topossible secondary structures. The high affinity Kd for binding to VEGFis shown for each ligand. One ligand in each family was selected forfurther analysis (gray box).

Of a total of 143 clones analyzed, 76 sequences differing by more thanone nucleotide were obtained. 44 of these sequences could be groupedinto three major families based on conserved primary structural motifs(Table 2). Sequences that may be grouped in minor families with five orfewer members and “orphan” sequences that were unique among the isolatesare shown in Table 6. Ligands containing the primary structural motifdefined by Families 1 and 2 arose in all three affinity selections.Similarities between the conserved primary structures of both familiessuggest that they may also share similar secondary structures and/orthat they may interface with VEGF using similar contact regions. Membersof Family 2 share the possibility of forming a short basepaired stemenclosing the conserved sequence motif in a large “loop” (underlined inTable 2). With the exception of the closing AXU basepair, the sequenceidentity of bases in the putative stem regions is not conserved. Such“co-variation” of bases that conserves secondary rather than primarystructure supports the existence of the putative stem and suggests thatthis structure may be important for the high affinity conformation ofthis family of VEGF ligands. No similarly conserved basepairinginteractions were detected among Family 1 sequences. A third family ofligands arose only in the selections performed in TBSMC (Family 3, Table2). In addition to a highly conserved primary structure motif, in allmembers of this family, sequences 3′ of the conserved region sharebasepairing complementarity to nucleotides in the 5′ fixed region(underlined in Table 2). Since, for most of the ligands, the bases onthe 5′ side of the putative stem cannot be said to covary with theirbasepairing partners, this observation is less predictive of a commonsecondary structure; nevertheless, our initial guess for a minimal highaffinity sequence derived from this family (described below) was guidedby the strong conservation of this motif. The affinities of theindividual RNA ligands for VEGF were estimated based on a singledetermination of the K_(D) for their interaction. With few exceptions,the ligands showed very high affinity for the growth factor, with K_(D)sbetween 5 and 50 pM.

Minimal Ligands

The shared primary and secondary structural motifs that define eachsequence family hint at the minimal sequence elements required for highaffinity binding to VEGF. Nested truncations of a representative ligandfrom each family (indicated by gray boxes in Table 2) were produced bychemical synthesis and their relative affinities for VEGF weredetermined (Table 3). Truncated versions of ligands VP30.22, VP30.2 andVT30.44 were prepared by chemical synthesis and their affinities forVEGF were determined as described in Example 3. Initial truncations(t22, t2, t44) were further refined by synthesis of oligonucleotideswith additional bases lacking from the 5′ and/or 3′ ends. In order toinitiate the chemical synthesis, the 3′-most nucleotide of several ofthe ligands was modified either by substitution of 2′-OH-cytidine for2′-F-cytidine (underlined) or by addition of a 3′-3′-linkeddeoxythymidine “cap” (asterisks). The length of each oligonucleotide(minus the cap) and its high affinity K_(D) for binding to VEGF areshown.

An initial prediction for the minimal sequence from clone VP30.22(Family 1) was made by mapping the ends of a purified, affinity-selectedfragment of the full-length ligand (see Example 3). This 29 nucleotidemolecule (“t22”) showed an approximately three-fold loss in bindingaffinity for VEGF relative to the full length ligand. Further truncationat the 3′ end of this molecule caused a precipitous loss in affinity butup to 6 additional nucleotides could be removed from the 5′ end withlittle or no consequence (Table 3). For clone VP30.2 from Family 2 andclone VT30.44 from Family 3, truncated ligands “t2” and “t44” weresynthesized that encompassed the putative five basepair stem and all ofthe conserved sequence motif. Both truncated ligands retained nearly allof the binding activity of the full length molecule. Further truncationby deleting one putative basepair at a time (one nucleotide from eachend of the ligand) caused a gradual loss in affinity. Thus, for thesesequences, truncations based on possible secondary structures predictedvery well the minimal high affinity ligand, and further supports thehypothesis that the putative stems contribute to the high affinityconformation of these ligands.

2′-OMe Modification

Substitution at the 2′-OH positions of RNA oligonucleotides by 2′OMe hasbeen observed to improve their stability against nucleases present inrat urine as well as in other biological fluids. Stabilization ofoligonucleotides to nucleases is likely to be critical to their successas therapeutic or diagnostic agents. Unfortunately, 2′-OMe-modifiednucleoside triphosphates are not generally accepted as substrates by RNApolymerases under standard reaction conditions. However, 2′-OMe purinesmay be introduced into a specific oligonucleotide by chemical synthesis.It has been observed that some high affinity 2′-OH purine RNA ligandswill accept a surprisingly high percentage of 2′-OMe purinesubstitutions with little loss of affinity for the target protein. Toidentify those purine positions for which 2′-OMe substitution iscompatible with high affinity binding to VEGF, several syntheses ofligands t2, t22 and t44 were prepared in which five or six purines at atime were partially substituted with the modified nucleotide (describedin Example 3). Affinity selection of each partially substituted librarywas used to isolate those molecules that retained substantial affinityfor VEGF. In such an affinity selected pool, positions that do nottolerate substitution are biased for 2′-OH and thus show highersensitivity to hydrolysis by alkali relative to the same position in theunselected library. 5′-radiolabeled unselected and affinity selectedpools were partially hydolysed by alkali and the products were displayedon a high resolution polyacrylamide gel. In ligand t22, G10 and A12showed substantial bias for 2′OH in the affinity selected pool, as didA6 and G21 in ligand t2, and A5 and A6 in ligand t44. While theforegoing analysis identifies those positions that are likely todisallow substitution with 2′OMe nucleotides, one cannot predict fromthese data how simultaneous modification of all other purines willaffect binding affinity. In fact, ligand t22, synthesized with all2′-OMe-purines except G10, A12 and G22 (which showed a marginalpreference for 2′-OH), bound to VEGF with an affinity equal to if notbetter than the all 2′-OH-purine sequence (Table 4).

Truncated oligonucleotides (t22, t2, and t44) were chemicallysynthesized with all but one, two or three purine positions substitutedwith 2′-OMe-purines. The remaining 2′-OH-purines are indicated in eachligand name and are shown in bold in the ligand sequence. KD_(S) for thebinding of each substituted ligand to VEGF are shown. Furthersubstitution at G22 had little effect on binding to VEGF, butincorporation of 2′-OMe at G10 or A12, as predicted, was detrimental tobinding affinity. Similarly, ligands t2 and t44 tolerated2′-OMe-substitution at all but two purines with a three- to four-foldimpact on the affinity of the Nucleic Acid Ligand for VEGF (Table 4).

Binding Affinities and Rate Constants for Substituted Truncates

In the hope of identifying highly 2′-substituted VEGF Nucleic AcidLigands of minimal length, all 2′-OMe-substitutions that did notdramatically decrease binding were incorporated into truncated ligandst22c, t2a, and t44a (see Table 3). 2′OH nucleotides are indicated inbold, and 2′OMe nucleotides are indicated in plain text. The resultantNucleic Acid Ligands, t22-OMe and t44-OMe, bound to VEGF with KDS of 67pM and 49 pM, respectively, while ligand t2OMe bound with a KD ofapproximately 140 pM (Table 5). These KDS compare favorably with that ofNX-213 (K_(D)=140 pM), a 2′-NH₂-pyrimidine-, 2′-OMe-purine-substitutedoligonucleotide inhibitor of VEGF described previously (see U.S. patentapplication Ser. No. 08/447,169, which is incorporated herein byreference). Each of the truncated 2′-OMe-substituted oligonucleotideswas found to compete with NX-213 and with one another for binding toVEGF.

Dissociation rate constants (k_(d)) were determined for each of thethree 2′-OMe-substituted ligands by following the loss of a preformedcomplex between radiolabeled ligand and VEGF upon the addition of alarge excess of unlabeled ligand. Ligand t22-OMe showed the fastest rateof dissociation with a half life of approximately 60 seconds. Ligandst2-OMe and t44-OMe showed slightly slower rates of dissociation withhalf lives on the order of 170 and 90 seconds, respectively. Associationrate constants (k_(a)), calculated from the equilibrium dissociationconstant and the dissociation rate constant (K_(D)=k_(d)/k_(a)), rangedfrom 3×10⁷ to 2×10⁸ M⁻¹ sec⁻¹ (Table 5). Such rapid rates of associationsuggest a near diffusion limited binding interaction between theseligands and VEGF, and are in line with the association rate constantsobserved for SELEX-derived Nucleic Acid Ligands to other targets.

Divalent Cation Dependence

Ligands in Families 1 and 2 were selected in the presence of magnesiumcations while Family 3 ligands were selected in a buffer containing bothmagnesium and calcium. Since divalent cations may contribute toRNA/protein interactions through nonspecific or specific stabilizationof high affinity RNA structures, we asked whether magnesium and/orcalcium were required for the high affinity binding of representativeligands to VEGF. The affinities of Nucleic Acid Ligands t22-OMe andt2-OMe (from Families 1 and 2, respectively) were unchanged in thepresence or absence of supplemental divalent cations or the chelatingagent EDTA (data not shown). However, Family 3 ligands, as representedby ligand t44-OMe, showed an absolute dependence on the presence ofcalcium for high affinity binding to VEGF. Binding was dramaticallyreduced (K_(D)>10⁻⁷) when divalent cations in the binding buffer werereplaced with EGTA. The addition of excess MgCl₂ todivalent-cation-depleted binding buffer gave no improvement in bindingaffinity, but CaCl₂, in two-fold molar excess over EGTA, fully restoredbinding activity. Identical binding behavior was observed for theunmodified ligand t44 (data not shown).

Protein Specificity

The oligonucleotides described here were selected based on theiraffinities for VEGF₁₆₅, the larger of two diffusible isoforms of thegrowth factor. VEGF₁₂₁, the smaller isoform, lacks one of the exons inVEGF₁₆₅ and, unlike the latter, does not bind to heparin. None of thethree truncated, 2′-OMe-substituted oligonucleotides bound with anymeasurable affinity to VEGF₁₂₁. Furthermore, the native structure ofVEGF₁₆₅ is essential for the binding of all three Nucleic Acid Ligands,as no binding is observed when the protein is reduced with DTT prior toincubation with the oligonucleotides.

VEGF is a highly conserved protein across species, the human VEGF₁₆₅ andmouse VEGF₁₆₄ isoforms showing 88% sequence identity. The truncated,2′-OMe-substituted ligands bound equally well to human and murine VEGF.However, no binding was observed for any of the ligands to homodimers ofPIGF, a placenta-derived protein that shares 53% sequence identity withVEGF across the conserved platelet derived growth factor-like domain.Heterodimers between VEGF and PIGF have recently been isolated from thesupernatants of both normal and tumor-derived cell lines, and suchheterodimers show activity in binding to one of two high affinity VEGFreceptors and in inducing responses in cultured endothelial cells. Thebiological relevance of VEGF/PIGF heterodimers is unknown. Substantialbinding, though with greatly reduced affinities, was observed withVEGF/PIGF heterodimers. These data may indicate that the Nucleic AcidLigands bind at or near the interface between the two subunits in adimer and that PIGF does not present all of the contact sites necessaryfor high affinity binding. Alternatively, the structure of the VEGFsubunit may be altered by participation in a heterodimer with PIGF withconsequent distortion of the Nucleic Acid Ligand binding surface.

Example 5 Synthesis of Phospholipid, Glycerol Amide Lipid, andPEG-modified VEGF Nucleic Acid Ligands

Three different formulations were used for the synthesis of variousLipophilic Compound/Nucleic Acid Ligand Complexes as follows:

1. C-18 Phosphoramidite for the Synthesis of PL Formulation

An outline for the preparation of C-18 phosphoramidite is shown inScheme 3. 1-Octadecanol was phosphorylated under standard condition.After work up the reaction mixture, the residue was purified on silicagel column with hexane:ethyl acetate:triethylamine (90:10:5) to offer21.5 g of pure product (57% yield).

II. Synthesis of Lipid Amide 1

This phosphoramidite, unlike the above PL, has amide linkages. Thestructure of the oligo resulting from conjugation of this lipid is shownbelow. Several experiments demonstrated that the high insolubility ofcompound 22 in organic solvents made NMR and MS characterization andfurther phosphitylation of compound 22 to DAG amidite 23 impossible,however, from the results for preparation of Lipid-spacer amidite(Scheme 3), we expected the phosphylation of compound 22 withchloro-(2-cyanoethoxy)-N,N-diisopropylamino-phosphine might go if themixture was refluxed. The approach to prepare the DAG amidite is shownin Scheme 4.

N,N′-Bis(stearoyl)-1,3-diamino-2-propanol (22) (2)

A solution of stearoyl chloride (6.789 g, 22.41 mmol) in ClCH₂CH₂Cl (50mL) was added dropwise to a solution of 1,3-diamino-2-hydroxy propane(1.0 g, 11.10 mmol) in ClCH₂CH₂Cl (100.0 mL) and TEA (2.896 g, 22.41mmol) with stirring at R.T. After finishing addition, the mixture washeated to 70° C. overnight, a clear solution was formed, and thesolution was cooled to R.T., filtered, and the solids were washed withCH₂Cl₂, CH₃OH, 5% NaHCO₃ and ethyl ether, and dried in vacuo to give 22(6.40 g, 93% yield) as white solids. ¹H NMR (pyridine-d₅; 60° C., δ,ppm): 3.82-3.78 (m, 1H), 2.37 (t, J=7.5 Hz, 4H), 1.81-1.76 (m, 4H),1.30-1.27 (m, 60H), 0.87 (t, J=5.7 Hz, 6H).

N,N′-Bis(stearoyl)-O-(diisopropylamino-2-cyanoethoxyphosphinyl)-1,3-diamino-2-propanol(23)

To compound 22 (5.80 g, 9.31 mmol), dried overnight in vacuo, was inanhydrous CH₂Cl₂ (150.0 mL) and N,N-diisopropylethylamine (4.2 mL, 18.62mmol) was injected. The mixture was cooled in an ice-water bath andchloro-(2-cyanoethoxy)-N,N-diisopropylamino-phosphine (8.6 mL, 0.47mmol) was injected. After stirring for 30 min, the mixture was heated at60° C. for 90 min. After cooling to R.T. insoluble materials werefiltered and the solution was washed with 5% NaHCO₃ and brine, driedover Na₂SO₄, and concentrated in vacuum. The crude product was purifiedby precipitation from CH₃CN to afford pure product (4.65 g, 61% yield)as white solids. ³¹P NMR (CDCl₃, ppm): 154.04.

1. Synthesis of DAG-Spacer Amidite, Lipid Amide 2

Hexa(ethylene glycol) was incorporated into the llipid amidite in orderto alleviate the insolubility of diamide compound 22, which is aimmediate intermediate to lipid amidite 23. An outline of thepreparation of lipid-spacer amidite 29 is shown in Scheme 5. Thecoupling step of compound 25 with 1,3-diamino-2-hydroxypropane andpotassium t-butoxide in THF did not go well, and the yield was onlyabout 20%. One attempt to improve yield was made by reacting 25 anddiamide 22, however, no desired product was detected.

(4,4′-Dimethoxytrityloxy)-hexaethylene glycol (24)

Hexa(ethylene glycol)(18.93 g, 67.05 mmol) was coevaporated withanhydrous pyridine (3×50 mL), dissolved in anhydrous pyridine (400 mL),and, after cooling to 0° C, DMTrCl (23.85 g, 70.40 mmol) in pyridine (50mL) was added dropwise during 30 min with stirring under Ar. Thereaction mixture was kept at R.T. overnight. The pyridine was removedunder high vacuum and the residue was dissolved in CH₂Cl₂, which waswashed with 5% NaHCO₃ and brine, dried over Na₂SO₄, and concentrated invacuum. The crude product was purified by wet-flash silica gel columnchromatography in a gradient of ethyl acetate, then CH₂Cl₂ and methanol(95/5) containing 0.5% TEA. The appropriate fractions were combined,evaporated, and dried in vacuum to give 24 (26.1 g, 66.6% yield) as alight yellow oil. ¹H NMR (DMSO-d₆; δ, ppm): 7.40 (d, J=7.2 Hz, 2H),7.33-7.24 (m, 7H), 6.89 (d, J=8.9 Hz, 4H), 4.61 (t, J=5.1 Hz, 1H), 3.73(s, 6H), 3.05 (m, 24H); ¹³C NMR (DMSO-d₆; δ, ppm): 158.02, 145.02,135.78, 129.67, 128.13, 127.71, 126,61, 113.14, 85.29, 72.33, 72.27,70.06, 69.87, 69.80, 69.75, 69.70, 62.84, 60.25, 60.19, 55.01.

(4,4′-Dimethoxytrityloxy)-hexaethylene glycol tosylate (25)

To an ice cooled solution (0° C.) of 24 in anhydrous pyridine (50 mL),was added a solution of toluene sulfonyl chloride in pyridine (30 mL).After 2 h at R. T., the solution was evaporated to a light yellow oil.The residue was taken-up in CH₂Cl₂ and washed with 5% NaHCO₃ and brine,dried over Na₂SO₄, filtered and evaporated in vacuo. The product waspurified by wet-flash silica gel chromatography, eluting with ethylacetate to give the product (4.08 g, 93% yield) as light yellow oil. ¹HNMR (DMSO-d₆; δ, ppm); 7.78 (d, J=8.3 Hz, 2H), 7.46 (d, J=8.1 Hz, 2H),7.40 (d, J=7.4 Hz, 2H), 7.32-7.23 (m, 7H), 6.88 (d, J=8.8 Hz, 4H), 4.09(t, J=4.3 Hz, 2H), 3.72 (s, 6H), 3.06 (m, 22H), 2.40 (s, 3H); ¹³C NMR(DMSO-d₆; δ, ppm): 158.01, 145.01, 135.78, 132.38, 130.12, 129.67,128.12, 128.02, 127.80, 127.70, 127.62, 113.13.

2-(4,4′-Dimethoxytrityloxy)-hexaethylene glycol-1,3-diaminopropane (26)

A mixture of 1,3-diamino-2-hydroxypropane (747 mg, 8.28 mmol) andpotassium t-butoxide (2.78 g, 24.84 mmol) in anhydrous THF was heated to70° C. for 2 h and then cooled to R.T. Compound 25 (4.08 g, 5.25 mmol)in THF was injected, and the mixture was stirred at 70° C. overnightuntil TLC showed on more 25 was left. After the solution was cooled toR.T., THF was removed in vacuo, and 25 mL of CH₂Cl₂ and 25 mL water wereadded. The CH₂Cl₂ layer was separated, and the water later was extractedwith CH₂Cl₂. The CH₂Cl₂ solutions were combined, dried over Na₂SO₄ andevaporated under reduced pressure. The crude product (2.43 g) wasdirectly used for reaction without further purification. ¹H NMR(DMSO-d₆; δ, ppm): 7.41 (d, J=7.7 Hz, 2H), 7.32-7.21 (m, 7H), 6.87 (d,J=8.8 Hz, 4H), 3.73 (s, 6H), 3.52-3.40 (m, 24H), 3.17 (s, 1H), 3.07-3.02(m, 4H).

N,N′-Bis(stearoyl)-2-(4,4′-dimethoxytrityloxy)-hexaethyleneglycol-1,3-diaminopropane(27)

A solution of stearoyl chloride (3.363 g, 11.1 mmol) in ClCH₂CH₂Cl wasinjected to a solution of 26 in ClCH₂CH₂Cl and TEA (1.9 mL, 11.1 mmol)with stirring at R.T. The mixture was kept at R.T. for 2 h, then heatedto 70° C. overnight. After the solution was cooled to R.T., the solutionwas washed with 5% NaHCO₃ and brine, dried over Na₂SO₄, and concentratedin vacuum. The crude product was purified by wet-flash silica gel columnchromatography in a gradient of ethyl acetate and CH₂Cl₂ (50/50) andthen ethyl acetate and methanol (50/50). The second fraction wascollected, evaporated, and dried in vacuum to give 27 (640 mg) as alight yellow solid. ¹H NMR (DMSO-d₆; δ, ppm): 7.40 (d, J=7.2 Hz. 2H),7.37-7.20 (m, 7H), 6.74 (d, J=8.9 Hz, 4H), 3.71 (s, 6H), 3.63-3.51 (m,24H), 3.17 (s, 1H), 3.16-3.13 (m, 4H), 2.12 (t, J=7.3 Hz, 4H), 1.18 (m,60H), 0.80 (t, J=6.2 Hz, 6H).

N,N′-Bis(stearoyl)-2-hexaethylene glycol-1,3-diaminopropane (28)

A mixture of compound 27 (640 mg), 2.5% DCA solution in CH₂Cl₂ (5 mL)and trihexylsilane (2 mL) was stirred at R.T. until orange color turnedto pale color. After removal of CH₂Cl₂, the residue was repeatedlyprecipitated from hexane to give a light yellow solid (210 mg, 63%yield). ¹H NMR (CDCl₃, δ, ppm): 3.3.69-3.59 (m, 24H), 3.17 (s, 1H),3.06-3.01 (m, 4H), 2.21 (t, J=7.9 Hz, 4H), 1.18 (m, 60H), 0.81 (t, J=6.3Hz, 6H).

N,N′-Bis(stearoyl)-2-(diisopropylamino-2-cyanoethoxyphosphinyl-hexaethyleneglycol)-1,3-diaminopropane (29)

Compound 28 (210 mg, 0.237 mmol), dried overnight in vacuo, wasdissolved in anhydrous CH₂Cl₂ (5.0 mL) and N,N-diisopropylethylamine(218 μL, 1.25 mmol) was added. The solution was cooled in an ice-waterbath and chloro-(2-cyanoethoxy)-N,N-diisopropylamino-phosphine (106 μL,0.47 mmol) was injected. After stirring for 30 min, the reaction mixturewas diluted with CH₂Cl₂ and washed with 5% NaHCO₃ and brine, dried overNa₂SO₄, and concentrated in vacuum to afford compound 29 ³¹P NMR (CDCl₃,ppm): 154.04.

Conjugation of 20K or 40K PEG NHS ester to VEGF Nucleic Acid Ligands

General procedure: VEGF oligonucleotide was exchanged forTriethylammonium salt and lyophilysed. The crude oligonucleotide wasdissolved in 100 mM sodium borate buffer (pH 9) to 60 mg /mlconcentration. 2 Eq of PEG NHS ester (Shearwater Polymers, Inc.) wasdissolved in dry DMF (Ratio of borate:DMF 1:1), and the mixture waswarmed to dissolve the PEG NHS ester. The oligonucleotide solution wasquickly added to the PEG solution and the mixture was vigorously stirredat room temperature for 10 min. About 90% of the oligonucleotide becomesconjugated to PEG NHS ester. See FIGS. 1H and 1I.

Synthesis of Dimeric VEGF Nucleic Acid Ligands

The dimeric VEGF Nucleic Acid Ligands shown in FIGS. 1J, K, and L weremade as follows.

Synthesis of 1,3-Dipivalolyl-2-O-dimethoxy tritylglycerol 32

To a stirred pyridine solution of compound 31 (62 g of 70% pure product,200 mmol, in 200 ml of pyridine), prepared according to McGee et al.(1988, Synthetic Communication, 1651), was added dimethoxy tritylchloride (84 g, 240 mmol, 1.2-fold excess) and the reaction was allowedto stir at room temperature for 16 hours. The reaction mixture wasconcentrated under reduced pressure and the residue was taken in CH₂Cl₂,(1 L) washed with water, and dried (MgSO₄) and concentrated. The crudemixture (130 g) was used as such in the next reaction.

Synthesis of 2-O-dimethoxy trityl glycerol 33

A mixture of crude compound 33 (130 g), NaOMe (28 g) and methanol (900ml) was heated at 50° C. for 16 h. After the reaction was complete(TLC), the mixture was concentrated to dryness and the residue wasdissolved in water and CH₂Cl₂ (1:1). The organic layer was separated,and the aqueous layer was washed with saturated NH₄Cl, water and brineand dried (MgSO₄). Evaporation of the solvent afforded a gummy compound,which was purified by silica gel column using 1:1 hexane/ethyl acetatecontaining 2% TEA to afford compound 33 in 75% isolated yield. ¹H NMR(DMSO-d₆) 3.02-3.07 (m, 2H), 3.17-3.23 (m, 2H), 3.3-3.35 (m, 1H), 3,7(s, 6H), 4.26 (t, J=4.1 2H, D₂O exchangeable), 6.59-6.86 (m, 4H),7.17-7.68 (m, 9H).

Synthesis of Bisamidite 34

To an ice cold stirred solution of the alcohol 3 (16.2 g, 41.1868 mmol)in CH₂Cl₂ (125 ml) and diisopropyl ethylamine (58 ml, 320 mmol) wasadded phophitylating reagent (20.5 ml, 90.62 mmol) and the solution wasslowly warmed up to rt and stirred for 2 h at the same temperature. Thereaction mixture was slowly poured into crushed ice and extracted withCH₂Cl₂, washed with 5% NaHCl₃, water and brine and dried. Residueobtained after evaporation of the solvent was purified by silica gelcolumn chromatography using 1:1 Hexane/ethyl acetate containing 2% TEAto afford compound 34 in 70% yield. ¹H NMR DMSO-d₆ 1.03-1.12 (2d, 24H),2.69-2.75 (2t, 4H), 3.1-3.33 (m, 4H), 3.33-3.55 (m , 5H), 3.66-3.7 (m,4H), 3.72 (s, 6H), 6.83-6.89 (m, 4H), 7.19-7.48 (m, 9H). ³¹PD₃PO₄ as anexternal standard 153.64 & 153.39 (2S).

Preparation of VEGF Dimers

Synthesis of VEGF dimers was done on 8800 automated DNA/RNA synthesizer.NX31838 was prepared, where rA stands for adenosine, mG and mA standsfor 2′-O-methylguanosine and adenosine respectively and fC and fU standsfor 2′-deoxy-2′-fluorocytidine and 2′-fluorouridine respectively and[3′-3′] stands for a 3′,3′-internucleotidic linkage. The synthesis wascarried out at a 1 mmol scale on a Millipore 8800 automated synthesizerusing 5′-DMT-2′-O-methyl-N⁶-tert-butylphenoxyacetyl-adenosine,5′-DMT-2′-O-TBDMS-N²-tert-butyl-phenoxyacetylguanosine and5′-DMT-2′-O-TBDMS-N⁶-tert-butylphenoxyacetyl-adenosine3′-N,N-diisopropyl-(2-cyanoethyl)phosphoramidites and2′-deoxy-2′-fluoro-5′-DMT-N4-acetylcytidine and2′-deoxy-2′-fluoro-5′-DMT-uridine3′-N,N-diisopropyl-(2-cyanoethyl)-phosphoramidites. The synthesis cyclewas as follows. The activator formulations are described in Table 12.The syntheses were carried out using CPG support of 600 Å pore size,80-120 mesh, and 60-70 μmol/g loading with 5′-succinylthymidine. Thecoupling cycle is shown in Table 12.

Example 6 Pharmacokinetic Properties of Phospholipid (PL) and PEGModified VEGF Nucleic Acid Ligands

Of the Sequences shown in Table 2, Sequence VT30.44 was chosen forfurther study and was renamed as NX31838. The pharmacokinetic propertiesof VEGF Nucleic Acid Ligand NX31838 conjugated to 20 and 40K PEG, weredetermined in Sprague Dawley rats (see FIG. 1 for moleculardescriptions) (SEQ ID NOS:8 and 9). Similar studies were also carriedout on NX31838 conjugated to PL lipid as a liposomal formulation and asfree drug (see FIGS. 1H and 1 for molecular descriptions) (SEQ ID NOS:8and 9). In each study the oligonucleotide was diluted in PBS to asolution concentration of 1.0 mg/ml based on UV absorption at 260 mn andan extinction coefficient of 0.037 μg oligo/ml. In all studies, 9 ratsreceived 1.0 mg oligonucleotide/kg animal weight by bolus tail veininjection and plasma samples were taken at various times from 2 minutesto 24 hours. The plasma samples and quality control samples wereanalyzed using a hybridization assay. The hybridization assay utilized acapture oligonucleotide that contains a complementary sequence to the5′-end of the VEGF Nucleic Acid Ligand conjugated to an iron oxide (FeO)bead (FeO-spacer-3′-d (GCC TTA GTC ACT T-5′) (SEQ ID NO: 137) wherespacer=(dT)₈), and a detection oligonucleotide containing two biotinmolecules at the 5′-end (biotin-biotin-5′-d(spacer-CGG ATG TAT AAGCA-3′), where spacer=(dT)₈) (SEQ ID NO: 138). After incubation of thecapture and detect probes with a plasma sample containing VEGF NucleicAcid Ligand NX31838 the amount of the biotin oligonucleotide hybridizedto the bead was quantitated with the strepavidin linked alkalinephosphatase, using CSPD-Sapphire as the luminescent substrate.

Data for the plasma concentration of the free, PEG20K and PEG40K VEGFNucleic Acid Ligands (NX31838) (SEQ ID NOS:8 and 9) as a function oftime following bolus injection are summarized in FIG. 6. The 40K PEGconjugate was cleared with a monoexponential t_(1/2) of 360 minutes,while the 20K PEG version was cleared much more rapidly with 95% of theNucleic Acid Ligand being cleared with an alpha t_(1/2) of 49 minutesand 5% being cleared with a beta t_(1/2) of 192 minutes, indicating theapparent importance of size on clearance. Compared with thePEG-conjugated Nucleic Acid Ligands, the free (unconjugated) NX31838 wascleared from plasma very rapidly with a t_(1/2) of several minutes. Theplasma concentration of an oligonucleotide as a function of time can besignificantly increased by introducing appropriate functional groupsinto the oligonucleotide.

Data for the plasma concentration of PL lipid conjugated VEGF NucleicAcid Ligand (SEQ ID NO:5) formulated with and without liposomes as afunction of time following bolus injection are summarized in FIG. 7. Theliposomes were created as described in Example 7A by sonication in thepresence of Nucleic Acid Ligand and contain oligonucleotide on theinside as well as the outside. The liposomal formulation was clearedmuch more slowly than the free drug, beta t_(1/2) of 1161 minutes and131 minutes, respectively. The plasma concentration of anoligonucleotide as a function of time can be significantly increased byliposomal formulation.

Example 7 Preparation of NX31838 PL-Liposome Complex

A. Liposome Preparation by Filming

The lipids are combined at a ratio of 2 moles DSPC to 1 molecholesterol. NX 31838 PL, in water, is added to the lipids at a ratio of1:50 (w/w). The material is combined by solvating with a solution ofchloroform:methanol:water (1:3:1). The solvent is removed by rotaryevaporation leaving a heterogeneous film of NX 31838 PL co-mixed withthe lipids. The film is rehydrated to 50 mg/mL, based on the lipids, ina solution of 9% sucrose, buffered with 25 mM sodium phosphate at pH7.4. The solution is mixed vigorously, heated to 65° C. and theresultant white milk-like solution sonicated in 75 mL aliquots toassemble the lipids into unilamellar liposomes. The progress of liposomeformation is followed visually until the solution becomes opalescent andthen by particle sizing via dynamic light scattering using a particleanalyzer (Leeds & Northrup Model Microtrack UPA 150, Horsham, Pa.).Liposome size is in the range of 50 to 70 nm (by volume weightdistribution method).

B. Liposome Preparation by Passive Anchoring

scNX-278 (see FIG. 1C for molecular description) was tested to seewhether it would undergo spontaneous incorporation into pre-formed(“empty”) liposomes. Preliminary results using a DEAE assay (for removalof free Nucleic Acid Ligand/glycerol lipid complex) indicated twoimportant findings: 1) loading could be achieved; and, more importantly,2) essentially complete loading of the Nucleic Acid Ligand/glycerollipid complex was observed over 24 hours at room temperature. A moredetailed study to determine the effects of temperature upon loading wassubsequently undertaken. It was observed that temperature had a dramaticeffect on the rates of incorporation. Although complete loading could beachieved over 24 hours at room temperature, complete incorporation couldbe achieved in just minutes at elevated temperatures (67° C.). Thisproved to be a rapid and efficient method for incorporating Nucleic AcidLigand/Lipophilic Compound Complex into pre-formed liposomes.

Size-exclusion chromatography was then used to separate free scNX-278from the liposome-associated form. The preliminary work was conductedusing the loading of scNX-278 into “empty” 2:1 DSPC:cholesterolliposomes. Chromatograms were generated using a Superdex S-200 column at22° C. Over a 22 hour period, the gradual incorporation of the scNX-278into the empty liposome population was observed as a shift in the peakareas (data not shown). The results correlate well with the dataobtained from the DEAE assay.

Studies were also undertaken to determine whether additional scNX-278could be loaded into sonicated oligo-liposomes. A sonicated preparationof scNX-278 was prepared by co-dissolving oligo-lipid with lipid andco-sonicating the two together. The resulting liposomes showed completeincorporation of the scNX-278. This sonicated preparation was thensubjected to 2 separate rounds of passive anchoring with additional freescNX-278 to see whether more scNX-278 could be incorporatedsuccessfully. During the first round of passive anchoring, all of thefree scNX-278 was passively anchored into the liposomes after incubationfor 1 hour at 65° C. The second attempt at passive anchoring ofadditional scNX-278 resulted in incomplete loading.

The key finding from these experiments is that a Nucleic AcidLigand/Lipophilic Compound Complex could be passively anchored intosonicated oligo-liposomes at high concentrations, but that theliposome's capacity for absorbing additional Nucleic AcidLigand/Lipophilic Compound Complexes could be exceeded. After 2 roundsof passive loading (to approx. 3 mg lipid-oligo/50 mg lipid), theliposomes apparently reach their “capacity” to absorb additionaloligo-lipid since some free lipid-oligo remains. These data wereconfirmed by DEAE spin-column analysis (data not shown). The conclusionsthat can be drawn are: 1) sonicated liposomes possess additionalcapacity for incorporating Nucleic Acid Ligand/Lipophilic CompoundComplexes; and 2) 100% Nucleic Acid Ligand incorporation can be achievedvia sonication.

Subsequent studies were conducted on NX31838 PL (see FIG. 1E formolecular description). NX31838 is of significant interest because ithas improved pharnacokinetics (see Example 6) and biodistributionagainst VEGF targets when incorporated in liposomes. Several studieswere conducted to better understand the incorporation of NX31838 viapassive anchoring into liposomes.

Studies on the kinetics of NX31838 PL indicated that the passiveanchoring for this molecule was so rapid as to be considered impossibleto measure via any of the chromatography techniques known in theliterature (all which require a minimum of several minutes of run time).

In order to determine the orientation of the NX31838 PL molecule (i.e.,whether the Nucleic Acid Ligand component was projecting externally fromthe Liposome, or projecting into the Liposome aqueous center),externally introduced RNase was used to selectively cleave any of theNucleic Acid Ligand Component that was projecting externally from theLiposome. In the case of passively anchored NX31838 PL liposomes, all ofthe Nucleic Acid Ligand is exposed to RNAse I. No additional digestionwas observed following Triton X-100 treatment. These results indicatethat the passively loaded NX31838 PL is oriented such that the NucleicAcid Ligand Component is projecting externally from the Liposome. If thepassively anchored NX31838 PL liposomes are pre-digested with RNAse I,then run over a DEAE column approximately 99% of the Nucleic Acid Ligandis captured by the column, whereas if the same sample is run over DEAEbut without pre-incubation with RNAse I, nearly 100% of the oligo isable to pass through the column, unbound to the DEAE. Liposome protectsthe oligo from DEAE. The Liposome acts to protect the Nucleic AcidLigand Component from DEAE, since it associates with the Nucleic AcidLigand with high affinity, greatly reducing its exposure to the DEAEgroups.

Finally, as part of developing new methods to separate free Nucleic AcidLigand/Lipophilic Compound Complex from the liposome-anchored form, wedigested NX31838.05 PL with RNase I. The cleaved oligo could be easilyseparated using size exclusion chromatography (S-1000 resin) followingremoval of the lipid tail, whereas the intact Nucleic AcidLigand/Lipophilic Compound Complex co-eluted with liposomes underidentical conditions. This data indicates that the Nucleic AcidLigand/Lipophilic Compound Complex is probably forming a micelle whenfree in solution. This results in it co-eluting in the void volume ofthe column with the liposomes. Removal of the lipid tail allows it toenter the gel filtration media and hence be sized and storedappropriately.

Example 8 In Vivo Efficacy of VEGF Nucleic Acid Ligand Complexes—DermalVascular Permeability Assay

The ability of several different formulations of the NX31838 NucleicAcid Ligand to attenuate VEGF-induced changes in the permeability of thedermal vasculature (Miles Assay) was performed as previously described(Senger et al. (1986) Cancer Research 46:5629-5632) with minormodifications. Briefly, adult female guinea pigs (3/study) wereanesthetized with isoflurane and the hair on the dorsal and lateral backareas was removed with clippers. Evans Blue dye (2.5 mg/guinea pig) wasadministered intravenously. Injection solutions (PBS, VEGF, NX31838formulations, and anti-VEGF monoclonal antibody) were prepared 30 min inadvance, co-mixed where indicated, with final concentrations as shown.Each solution shown was then injected intradermally (duplicateinjections/guinea pig; 40 μl/site) in a randomized manner in a gridpattern drawn on the clippered area. Guinea pigs were allowed to recoverfrom anesthesia and were sacrificed by CO₂ exposure 30 min aftercompletion of the intradermal injections. The skin was then harvested,trimmed free of subcutis, and transilluminated. Images were thencaptured using a color CCD camera (Hitachi Denshi KP-50U, Japan) andImage-Pro Plus software (Version 3.1, Media Cybernetics, Silver Springs,Md.). Each skin sample was normalized for intensity with each injectionsite analyzed for optical density and the area involved.

Panels A-C from FIGS. 8A-C show the results of Nucleic Acid Ligandattenuation of VEGF-induced vascular leakage for NX31838-20K PEG,NX31838-40K PEG, NX31838-PL in liposomal preparation as described inExample 7A. All formulations were able to significantly reduce vascularleakage down to or near PBS control levels with concentrations as low as100 nM. At 30 nM the blocking effect of the Nucleic Acid Ligand waslost. The NX31838-PL liposomal formulation was not evaluated at thisconcentration but appeared to have reduced blocking activity at 100 nM.The anti-VEGF monoclonal antibody was also evaluated in this modelsystem and was likewise effective down through 100 nM with loss ofactivity at 30 nM. Thus, suggesting that in this model system thatNX31838 in the various formulations examined is equally effective asantibody in blocking one of the functional effects of VEGF protein.

Example 9 In Vivo Efficacy of VEGF Nucleic Acid Ligand Complexes—CornealPocket Model

VEGF Nucleic Acid Ligand (NX31838) formulations were tested in theirability to reduce VEGF-induced corneal angiogenesis in the normallyavascular rat cornea. Briefly, biopolymer (Hydron) pellets±VEGF protein(3 pmol) were prepared approximately 30 hr before by adding the proteinor carrier solution to 12% biopolymer in 95% ethanol. Adult,Sprague-Dawley rats (200-240 g) were anesthetized by intraperitonealinjection of ketamine HCl (50 mg/kg) and xylazine (10 mg/kg). The lefteye was then prepared by topical administration of tetracaine HCl forlocal anesthesia followed by application of dilute povidone-iodinesolution and subsequent rinsing with isotonic saline solution. Avertical partial thickness incision was made in the mid-cornea. Amid-stromal pocket was dissected caudally toward the lateral canthusextending to within 1.5 mm of the limbus. A pellet was then insertedinto and pushed to the caudal limit of the pocket. Residual air wasgently massaged out of the pocket. A drop of chloramphenicol ophthalmicsolution was then applied to the eye. The animal was rolled over and theprocedure repeated on the right eye with insertion of the same type ofpellet. Upon completion of pellet insertion in each eye, each animal wasthen administered either PBS (volume matched to Nucleic Acid Ligandformulation group) or Nucleic Acid Ligand (10 mg/kg) intravenously twicedaily as indicated. At 5 days, each animal was anesthetized andphotographs were taken using a 35 mm camera (Minolta X9) mounted on adissecting microscope (KAPS, Germany). Each eye was evaluated for theangiogenic response by measuring the maximum length of vessel growth(0-5), the density of vessel growth (1-4) adjacent to the implantedpellet, and the circumference of the eye with angiogenesis occurring(0-1). An angiogenic index was then determined as the product oflength * density * circumference.

The ability of Nucleic Acid Ligand formulations to block VEGF-inducedangiogenesis is seen in FIGS. 9A-C Despite being equally effective asthe other formulations in blocking vascular permeability changes, NX31838-20K PEG was ineffective at attenuating the angiogenic response inthe normally avascular cornea. However, both NX31838-40K PEG andliposomal NX31838-PL significantly reduced the level of angiogenesis by65-70%. It is presumed that these differences are attributable to therespective pharmacokinetic profiles of the Nucleic Acid Ligands.

Statistical Analysis: Groups in the Miles assay and corneal angiogenesismodels were compared using Rank ANOVA with Dunnett's comparisons.

Example 10 In Vivo Efficacy of VEGF Nucleic Acid Ligand in Tumor Models

Human Tumor Xenograft Model

The ability of the VEGF Nucleic Acid Ligand NX31838 40K PEG to affectsolid tumor growth was determined in a subcutaneous tumor model in nudemice. The A673 human rhabdomyosarcoma tumor cells were grown in tissueculture, harvested and 1×10⁷ viable cells were implanted subcutaneously,in nude mice, proximal to the axillary region of the flank. Treatmentwith test compounds was initiated 12 hours later, and continued for theduration of the experiment. Compounds were dosed intraperitoneally,twice daily at 10 and 40mg/kg. A negative control consisted of dosing ascrambled aptamer sequence, NX31917-40K PEG (See FIG. 1R for moleculardescription) at 40 mg/kg twice daily, and a positive control consistedof anti-VEGF antibody Mab.26503.11 (R & D Systems, Lot # LD03) dosed at100 μg/ mouse twice weekly. Both Nucleic Acid Ligand-treated groups, andthe antibody treated groups demonstrated a significant slowing of tumorgrowth relative to the scrambled sequence negative control group (FIG.11). The % Tumor Growth Inhibition (TGI), was determined to be 75% and80% for the 40mg/kg and 10 mg/kg BID groups and 83% for the monoclonalantibody treated group (Table 8). Since there appeared to be nosignificant difference between the 40mg/kg dose group and the 10 mg/kgdose group, no further dosing of the 40mg/kg group occurred after day14. As can be seen in FIG. 11, several days after termination of dosing,tumors grew rapidly and mimicked the growth rate of the negative controlgroup, while the 10 mg/kg Nucleic Acid Ligand group and the antibodytreated group continued to grow at a reduced rate.

Additional studies were performed using the same tumor model where newbatches of VEGF Nucleic Acid Ligand, NX31838 40K PEG (designatedNX31838.04 and NX31838.07) were compared, and also dose titrateddownward from 10 mg/kg BID, 3mg/kg BID and 1 mg/kg BID. The experimentalso included a once daily dose of 10 mg/kg, as well as a Liposomal formof the VEGF Nucleic Acid Ligand, NX31838 PL at 10 mg/kg BID. As can beseen in FIG. 12 and Table 9, the same degree of tumor growth inhibitionwas achieved in both experiments. Both batches of VEGF Nucleic AcidLigand were equivalent when compared on the twice daily dosing schedule,with TGI values of 61% and 70% for the old and new batch, respectively.In addition, it was determined that the once daily dosing (SID) was aseffective as the twice daily dosing. However, the titration scheme usedin this experiment failed to reach a no effect dose.

A third experiment was performed where further downward titration of theVEGF Nucleic Acid Ligand was able to demonstrate a dose responserelationship relative to tumor growth. In this experiment the VEGFNucleic Acid Ligand was titrated down, approaching a no effect dose of0.03 mg/kg. The relative tumor growth inhibition can be seen in FIG. 13and is summarized in Table 10.

In addition to the three unstaged tumor studies, a staged tumor studywas prepared where the tumors were allowed to establish and reach200+/−100 mm³ prior to initiation of treatment with the VEGF NucleicAcid Ligand. The dose groups of 10 mg/kg of NX31838 40K PEG and the 100μg twice weekly of mAb 26503 (R & D Systems) achieved 59% and 69% tumorgrowth inhibition respectively (FIG. 14, Table 11). These collectivestudies demonstrate that the VEGF Nucleic Acid Ligand is able to slowthe A673 tumor from establishing as well as inhibiting tumor growth oncetumors have established.

Kaposi's Sarcoma Model

The effect of NX 31838-40K PEG on the subcutaneous growth of Kaposi'sSarcoma cell line KSY-1 in nude mice was also examined. KSY-1 cells areunique among tumor cell lines in that they can be inhibited in cultureby VEGF antagonists. KSY-1 cells were grown in culture, pooled andinjected subcutaneously (2×10⁷ cells/mouse) in the hind flank of mice.Three groups of mice (4 mice per group) were treated by intraperitonealinjections every 12 hours with either 30 mg/kg of NX31838-40 K PEG, 30mg/kg NX31917-40 K PEG (see FIG. 1R for molecular description) or PBSfor the duration of the experiment. Treatment was initiated one dayafter tumor cell implantation. While tumor growth in the PBS-treated andNX31917-40 K PEG-treated groups was comparable, considerable inhibitionof tumor growth was observed in the NX31838-40K PEG-treated group (FIG.16). NX31838-40K PEG inhibited the growth of KSY-1 tumors by 65%(compared with the PBS-treated group) or by 69% (compared with theNX31917-40 K PEG-treated group) at the time the experiment wasterminated (day 22).

Example 11 Intravitreal Pharmacokinetics of VEGF Nucleic Acid LigandNX31838+40KPEG in Rabbits

New Zealand White rabbits were treated with VEGF Nucleic Acid LigandNX31838 conjugated to 40 mPEG by intravitreal administration at a doseof 0.5 mg/eye. 40K PEG was conjugated to the VEGF Nucleic Acid Ligand asdescribed in Example 5, and the resulting complex is as shown in FIG. 1H(SEQ ID NO:8). Rabbits received intravitreal injection of NX31838-40KPEG in each eye. The time between doses for a given animal did notexceed 15 minutes. Blood and vitreous samples were collected asspecified in Table 7.

Analysis of plasma and vitreous samples were carried out by the use of adouble hybridization assay. In this assay, two hybridization probes areused, a capture probe attached to wells of 96 well plates, and abiotinylated detect probe. The capture probe forms a hybrid with the 5′end of the Nucleic Acid Ligand. This assay is highly specific andsensitive to full length Nucleic Acid Ligand to yield a positive signal.The current limit of quantitation is approximately 2 fmoles in 5 μofplasma.

TABLE 1 Summary of VEGF Nucleic Acid Ligand pharmacokinetic parametersafter i. v. bolus administration in Sprague Dawley rats determined fromthe data shown in FIG. 15 (compartmental analysis). Parameter NX213NX278 NX278-L Total AUC (μg * min/ml) 147 202 531 C, t = 0 min (μg/ml)14.59 23.16 16.95 C, t = 2 min (μg/ml) 15.31 14.08 15.74 αt_(1/2) (min)7 3 13 βt_(1/2) (min) 49 67 113 Clearance (ml/kg/min) 6.80 4.95 1.88V_(ss) (ml/kg) 72 251 152

TABLE 2 2′-F-pyrimidine ligands to VEGF₁₆₅ Ligand Sequence of variableregion K_(d) SEQ. ID NO: (frequency) 5′-gggaggacgaugcgg [variableregion] cagacgacucgcccga-3′ (pM) 14 & 139 Family 1 VP30.7 gcggggAAGAAUUGG UCAUCGUCGUCUCCGCCUCCC 3000 15 VP30.12 gcggAAUACG GAAGAAUUGGAUACAUAUGCUCGU 7 16 VP30.13 (7) ugcggGAUAACA GAAGAAUUGG UGAACAACGUGGU 1017 VP30.16 AUGAUCGCGUAG GAAGUAUUGG AAGGCCCU 6 18 VP30.19 gcggCACUUUAGAAGAAUUGA AUUUCCCGCUGGU 9 19 VP30.22 (6) gcggUAG GAAGAAUUGGAAGCGCAUUUUCCUCGY 20 20 VP30.25 cggCGGGAUUUUG GAAGAAUUGG AUAUUGGCCU 2021 VP30.26 (2) gcggCGGYACUUUG GAAGAAUUGA AUUUCCCGCU 10 22 VP30.27 gcggggAAGAAUUGG AUAUAUCGUUCACCCCCACCU 400 23 VP30.40 ugcgAAACG GAAGAAUUGGAUACGCAAGCACGUU 6 24 VP30.41 gcggUAG GAAGUAUUGU AAGCGCCUCGUUUUCGC 7 25VP30.51 (2) gcggAGUUUUG GAAGAAUUGG AUGUUCCGAUCGU 90 26 VP30.54gcggAAGAAACG GAAGAAUUGG AGACACGCUCGU 10 27 VP40.4 (5) gggaggacgaugcggGAAGAAUUGA UGUUGUAUUGUCCUUCCGAUUUCCUGCCGU 200 28 VP40.43ggaggacgaugcggACA GAAGAAUUGG GCUUCGCAUUAUCCUCUGUCAGCCGC 30 29 VP40.53ugcggUGAGAGAAACG GAAGAAUUGG AUACGAUACUCAUCGCGCU 8 30 VT30.4augcggCUUAAGUUUUG GAAGAAUUGA AUACUGGGU 20 31 VT30.7 gcggUAACCAGUGGAAGAAUUGG CUGCUAUCCU 10 32 VT30.10 augcggAACG GAAGAAUUGGAUACGUAGCAUGCGU 2 33 VT30.13 ugcggCAGGAUUUUG GAAGAAUUGG AUAUUGGCCGca 1034 VT30.20 gaugcggAAACG GAAGAAUUGG AUACCGCUACGUGUU 4 35 VT30.52 ugcggggAAGAAUUGA GCAUUCCUUCUCCUUGUGCCU 9000 36 VT30.53 gaugcggAGCUAACGGAAGAAUUGG AAACAACCGCGUc 10 37 Family 2 VP30.2 (5) ggYGA ACCGA UGGAA UUUUUGGACGC UCGCCU 10 38 VP30.5 (4) gAYCA ACCGA UUGAC GUUA UGGGACGC UGGUc 839 VP30.31 (5) gcggUA ACCGA UUGAA CUUC UUGGACGC UACCGU 6 40 VP30.43 ggUAACCGAA UUGAA GUUA UUGGACGC UACCU 5 41 VP40.9 gGAGCAGA ACCGA UAGAA GAAUUGGACGC UCAGCUCCGGGU 30 42 VP40.14 GUACCAGAAUGAGCA ACCGA AUGAA GAACUGGACGC UGCUca 8 43 VP40.17 ugcggUGA ACCGA UGGAA UCGC UUGGACGCUCAUCGCACGUUGCU 10 44 VT30.9 (6) ggUCA ACCGG UUGAA UAU UUGGUCGC UGACCU30 45 Family 3 VT30.1 (2) gacgaugcgg A ACUA GUGAAUGCUU AUA CGACCGUGUUGUc 10 46 VT30.2 gcgg AUCA GUGAAUGCUU AUA GA CCGCCUCCGU 2 47VT30.3 (7) gaugcgg AGA AUCA GUGAAUGCUU AUA AAUC UCGYGUc 5 48 VT30.11gaugcgg A AUCA GUGAAUGCUU AUA GCUC CCGCGUCCU 4 49 VT30.15 gcgg A ACCAGUGAAUGCUU AUA AGA CUGCUCGU 3 50 VT30.21 cgaugcgg AUCA GUGAAUGCUU AUA GACCGUAUUGCGU 6 51 VT30.28 gaugcgg AGA AUCA GUGAAUGCUU AUA AACC UCGUGUc 6052 VT30.29 augcggA AUCA GUGAAUGCUU AUA GC UCCGCGUGGU 10 53 VT30.35 cggACCA GUGAAUGCUU AUA AGCCCA UCGACCU N.D. 54 VT30.41 gaugcgg CAGGGUGAAUGCCA AUG UACUUU UCGCGUc 40 55 VT30.42 gacgaugcggA AUCA GUGAAUGCUUAUA GC UCCACGUCGUc N.D. 56 VT30.44 gcggA AUCA GUGAAUGCUU AUA CAUCCGCUCGGU 10 57 VT30.54 gcggG ACUAG GUGAAUGCCA AUA UUCUUC UCCGU 10 58

TABLE 3 Length K_(D) SEQ ID Ligand Sequence (nts) (pM) NO: t22GACGAUGCGGUAGGAAGAAUUGGAAGCGC* 29 70 59 t22aGACGAUGCGGUAGGAAGAAUUGGAAGCG 28 3000 60 t22bACGAUGCGGUAGGAAGAAUUGGAAGCGC 28 80 61 t22c GCGGUAGGAAGAAUUGGAAGCGC 23 9062 t22d CGGUAGGAAGAAUUGGAAGCGC 22 100 63 t22e GGUAGGAAGAAUUGGAAGCGC* 21200 64 t22f GUAGGAAGAAUUGGAAGCGC* 20 >100,000 65 t2GGCGAACCGAUGGAAUUUUUGGACGCUCGCC* 31 20 66 t2aGCGAACCGAUGGAAUUUUUGGACGCUCGC 29 40 67 t2b CGAACCGAUGGAAUUUUUGGACGCUCG27 100 68 t2c GAACCGAUGGAAUUUUUGGACGCUC* 25 200 69 t2dAACCGAUGGAAUUUUUGGACGCU* 23 20,000 70 t2e ACCGAUGGAAUUUUUGGACGC*21 >100,000 71 t44 GCGGAAUCAGUGAAUGCUUAUACAUCCGC* 29 10 72 t44aCGGAAUCAGUGAAUGCUUAUACAUCCG 27 10 73 t44b GGAAUCAGUGAAUGCUUAUACAUCC 2560 74 t44c GAAUCAGUGAAUGCUUAUACAUC* 23 2000 75 t44dAAUCAGUGAAUGCUUAUACAU* 21 >100,000 76 t44e AUCAGUGAAUGCUUAUACA*19 >100,000 77

TABLE 4 Effect of 2′-OMe-purine substitutions on affinity for VEGF.K_(D) SEQ ID Ligand Sequence (pM) NO: t22OMe (OH-10,12,22)GACGAUGCGGUAGGAAGAAUUGGAAGCGC 10 78 t22OMe (OH-10,12)GACGAUGCGGUAGGAAGAAUUGGAAGCGC 20 79 t22OMe (OH-10,22)GACGAUGCGGUAGGAAGAAUUGGAAGCGC 4,000 80 t22OMe (OH-12,22)GACGAUGCGGUAGGAAGAAUUGGAAGCGC 90 81 t2OMe (OH-6,21)GGCGAACCGAUGGAAUUUUUGGACGCUCGCC 60 82 t2OMe (OH-6)GGCGAACCGAUGGAAUUUUUGGACGCUCGCC 500 83 t2OMe (OH-21)GGCGAACCGAUGGAAUUUUUGGACGCUCGCC 20,000 84 t44OMe (OH-5,6)GCGGAAUCAGUGAAUGCUUAUACAUCCGC 40 85 t44OMe (OH-5)GCGGAAUCAGUGAAUGCUUAUACAUCCGC >100,000 86 t44OMe (OH-6)GCGGAAUCAGUGAAUGCUUAUACAUCCGC >100,000 87

TABLE 5 K_(D)(S.d.) k_(d)(s.d.) k_(a) SEQ Ligand Sequence (pM) (sec⁻¹)(M⁻¹sec⁻¹) ID NO: t22OMe GCGGUAGGAAGAAUUGGAAGCGC 67 (36) 0.012 (0.004)1.8 × 10⁸ 88 t2OMe GCGAACCGAUGGAAUUUUUGGACGCUCGC 140 (50) 0.0042 (0.002)3.0 × 10⁷ 89 t44OMe CGGAAUCAGUGAAUGCUUAUACAUCCG 51 (ll) 0.0074 (0.002)1.5 × 10⁸ 90

TABLE 6 Additional 2′-F-pyrimidine ligands to VEGF₁₆₅. Ligand Sequenceof variable region Kd SEQ. ID NO: (frequency) 5′-gggaggacgaugcgg[variable region] cagacgacucgcccga-3′ (Pm) 14 and 139 VP30.1UCUUUGAGUUUUUGCCAACGGUUUUCGCU 32,000 91 VP30.6AACGGAAUUCUUGGAUACACACCUCGUCCU 20 92 VP30.11UCAGGAACGGAAUUUUUGGAGACACGCCCU 25 93 VP30.14ACUGGGAGAAUCCGAAAAACCUUCACGCGU 25 94 VP30.18AUCCAUCAUUUAACCGUUUGCUCUCCCCCU 27 95 VP30.20 (3)UUGAUCGGACGUUAGUCAUUUCCCGAUCGU 57 96 VP30.23GAGCUUGAAGUUUCAGUAUUGGCACAACCU 63 97 VP30.29CGCCACUUUGGAAGUUAUUGAAUUUCGCGU 7 98 VP30.35UGAAUGAGCUGACGACCCUGAAUUGCUCGU 6 99 VP30.48GAGCUUGAAGUUUCGGUAUUGGCACAACCU >10,000 100 VP30.58CAACUAUUCGUUGAUGUUUCCGUGAGCCGU 6 101 VP30.61GAGCUUGAAGUUUCAGUACUGGCACAACCU 43 102 VP30.63AACCAAUAGAGAUCUUCGGCUGCCCCGCGU 16 103 VP30.65AAAACGCUUUUCUUGGCCCCCUCGUUGCGC 33 104 VP30.67UUAACGGAAUUCUUGGAUACAUAGCAUGGU 24 105 VP40.1CAAAGUUUGAGUUGAUCUGAUACGUUUCAGUAUUGGCGU N.D. 106 VP40.2 (5)AUCUGUGAACUGGGUUUUUGCCGACGGUUACGCUUUUGCU 35 107 VP40.3 (5)CAAAAGUUUGAGUUGAUCUGAUACGUUUCAGUAUUGGCGU 2,000 108 VP40.5UUGAUCGAGGUUCUAAAGCCUAUUUCCUGACUUUCUCCCC 19 109 VP40.10AUCUGUGAACUGGGUUUUGCCGACGGUUACGCUUUUGCU N.D. 110 VP40.11 (6)AAGGAAGAUGUUGAUCGUUUGACGUGAUGUGGAUCCGCGU 980 111 VP40.18UAGUAAGUUAUUGAAAGCGCAUCUCUAUCAACUCUCGGCC 12 112 VP40.20UACUUUCUUCUUUCUUUGCCUUUCUUUUUCUUUUACGCCU N.D. 113 VP40.21CAGUUAAUUAAUUUGAGUUGUGAUGUGUGUCGUUAUGGGU >100,000 114 VP40.24GAUGCUGAGUGAGGAAGUCUGAUUGUUGCAGUAUUGGCGU 5,000 115 VP40.25AAUGGAAUUUGAGUCGAUCUAGAAUGCGUCGUAUGGGCU 740 116 VP40.26ACUCAACUGGACGCUAUGUUGACGGUUAUCGCUUUUGGGU 13 117 VP40.36CAGGUUCAGAAUUGGCAGUCGCAUUGAUCUUUUUCACCGC 1,300 118 VP40.37CAAAAGUUUGAGUUGAUCUGAUACGUUUCCAGUAUUGGCGU N.D. 119 VP40.39CAGUUAAUUAACUUGAGUUGUGAUGUGUGUCGUUAUGGGU 73,000 120 VP40.41CAAAUUCAAGGUCGAGUUAUGCGUAGAUGUGGCUCCUGUG 11,000 121

TABLE 8 Anti-Tumor Efficacy of VEGF Nucleic Acid Ligand (NX31838) in theA673 Nude Mouse Xenograph Model % Tumor Growth Inhibition (TGI) MeanTumor % Treatment Volume Day TGI Scrambled Nucleic Acid Ligand 40 mg/kgBID 2823 13 0  VEGF Nucleic Acid Ligand 40 mg/kg BID  710 13 74.8 VEGFNucleic Acid Ligand 10 mg/kg BID  565 13 80.0 Anti-VEGF mAb 2× weekly 489 13 82.7 % TGI = 100 (1 − W_(t)/W_(c)); W_(t) is the mean tumorvolume of the treated group at time x; W_(c) is the mean tumor volume ofthe control group at time x 42% TGI or greater is significant Day to GDDay to Tumor Growth Delay 1000 mm³ (t_(t) − t_(c)) 1500 mm³ GD ScrambledNucleic Acid Ligand  8.5 0 10  0 40 mg/kg BID VEGF Nucleic Acid Ligand14.5  6** — — 40 mg/kg BID VEGF Nucleic Acid Ligand 17     8.5 20 10 10mg/kg BID Anti-VEGF mAb 2× weekly 18     9.5 21 11 **note: dosing forVEGF Nucleic Acid Ligand @ 40 mg/kg was terminated on day 14

TABLE 9 Anti-Tumor Efficacy of VEGF Nucleic Acid Ligand (NX31838) in theA673 Nude Mouse Xenograph Model % Tumor Growth Inhibition (TGI) MeanTumor Volume # % Treatment mean (± SD) Animals Day TGI PBS control 2357(± 1361) 8 16  0 VEGF NX31838.04 930 (± 312) 7 16 61 10 mg/kg BID VEGFNX31838.04 1135 (± 364)  7 16 52 3 mg/kg BID VEGF NX31838.04 1045 (±265)  8 16 56 1 mg/kg BID VEGF NX31838.04 713 (± 206) 6 16 70 10 mg/kgSID VEGF NX31838.07 570 (± 273) 6 16 76 10 mg/kg BID VEGF NX31838PL 555(± 174) 8 16 76 10 mg/kg BID % TGI = 100 (1 − W_(t)/W_(c)); W_(t) is themean tumor volume of the treated group at time x; W_(c) is the meantumor volume of the control group at time x 42% TGI or greater issignificant

TABLE 10 Anti-tumor Efficacy of VEGF Nucleic Acid Ligand (NX31838) 40KPEG in Nude Mouse Xenograph Model % Tumor Growth Inhibition (TGI) MeanTumor Volume # % Treatment Mean (± SD) Animals Day TGI PBS Control 3446(± 1522) 8 14  0 NX31838 10 mg/kg 540 (± 122) 8 14 84 NX31838 3 mg/kg795 (± 403) 7 14 77 NX31838 0.3 mg/kg 1261 (± 337)  8 14 63 NX31838 0.03mg/kg 1773 (± 785)  8 14 49 % TGI = 100 (1 − W_(t)/W_(c)); W_(t) is themean tumor volume of the treated group at time x; W_(c) is the meantumor volume of the control group at time x 42% TGI or greater issignificant

TABLE 11 Anti-Tumor Efficacy of VEGF Nucleic Acid Ligand (NX31838) vsAnti-VEGF MAb in a Growth Staged A673 Xenograph Model % Tumor GrowthInhibition (TGI) Mean Tumor Volume # % Treatment Mean (± SD) Animals DayTGI PBS Control 3082 (± 1198) 8 12  0 NX31838 10 mg/kg 1278 (± 543)  812 59 Anti-VEGF Mab 100 μg Twice 959 (± 359) 8 12 69 weekly % TGI = 100(1 − W_(t)/W_(c)); W_(t) is the mean tumor volume of the treated groupat time x; W_(c) is the mean tumor volume of the control group at time x42% TGI or greater is significant Tumor Growth Delay (TGD) Days to TGDDays to TGD Treatment 1000 mm³ (t_(t) − t_(c)) 2000 mm³ (t_(t) − t_(c))PBS Control 6.3  9.5 NX31838 10 mg/kg 10   3.7 15.5 6   Anti-VEGF mAb100 μg 12.3  6   18.3 8.8 Twice weekly

TABLE 12 Automated synthesis cycle for the preparation of NX31838 Equiv-Step Reagents alents* Reaction Time Detritylation Dichloroacetic acid in250 15 min CH₂Cl₂ (3% v/v) Coupling Nucleoside phosphoramidite  2 20 minfor fC & (0.2M in CH₃CN) fU and 30 min for all others 1.0 M DCI  8**Oxidation 0.05 M I₂ in 2:1  5.2 2 pyridine:water Capping Cap A: 1:1:8 32 1 Ac₂O:2,6-lutidine:THF Cap B: 16% NMI in THF *Equivalents are basedon the moles of CPG-bound 3′-terminal nucleoside. **Activatorequivalents are based on moles of nucleoside phosphoramidite.

General Procedure for the Synthesis of Dimers

All other phosphoramidites were coupled by the same manner as mentionedabove, except the glycerol bis amidite

Equiv- Step Reagents alents* Reaction Time Detritylation Dichloroaceticacid in 250 15 min CH₂Cl₂ (3% v/v) Coupling Nucleoside phosphoramidite0.75 eq 2 × 20 min (Coupled (0.04M in CH₃CN) per twice) coupling 1.0 MDCI 16** Oxidation 0.05 M I₂ in 2:1 5.2 2 pyridine:water Capping Cap A:1:1:8 32  1 Ac₂O:2,6-lutidine:THF Cap B: 16% NMI in THF

139 33 nucleic acid single linear RNA Nucleotides at positions 1-4 and29-33 are bound by a phosphorothioate bond Nucleotides at positions 5,13, 16, 17, 20, 23-26, and 28 are 2′-OMethyl (2′-OMe) modifiedNucleotides at positions 6-9, 12, 15, 19, 21, 22 and 27 are 2′-amino(2′-NH2) modified 1 TTTTACCCUG AUGGUAGACG CCGGGGUGTT TTT 33 33 nucleicacid single linear RNA Nucleotides at positions 1-4 and 29-33 are boundby a phosphorothioate bond Nucleotides at positions 5, 13, 16, 17, 20,23-26, and 28 are 2′-OMethyl (2′-OMe) modified Nucleotides at positions6-9, 12, 15, 19, 21, 22 and 27 are 2′-amino (2′-NH2) modified 2TTTTACCCUG AUGGUAGACG CCGGGGUGTT TTT 33 33 nucleic acid single linearRNA Nucleotides at positions 1-4 and 29-33 are bound by aphosphorothiate bond Nucleotides at positions 5, 8-9, 11, 14, 16, 18,23, 26, and 28 are 2′-OMethyl (2′OMe) modified Nucleotides at positions6-7, 10, 12, 17, 21-22, 24-25, and 27 are 2′-amino (2′-NH2) modified 3TTTTGUCGGU ACGGAGUGGA CCGUCACGTT TTT 33 33 nucleic acid single linearRNA Nucleotides at positions 1-4 and 29-33 are bound by aphosphorothiate bond Nucleotides at positions 5, 8-9, 11, 14, 16, 18,23, 26, and 28 are 2′-OMethyl (2′OMe) modified Nucleotides at positions6-7, 10, 12, 17, 21-22, 24-25, and 27 are 2′-amino (2′-NH2) modified 4TTTTGUCGGU ACGGAGUGGA CCGUCACGTT TTT 33 28 nucleic acid single linearRNA Nucleotides at positions 1, 6-7, 10, 14, 16-18, 20, 22, and 24-26are 2′-fluoro (2′-F) modified Nucleotides at positions 2-3, 8-9, 11-13,15, 19, 21, 23, and 27 are 2′-OMethyl (2′-OMe) modified T in position 28is a 3′-3′-linked deoxythymidine 5 CGGAAUCAGU GAAUGCUUAU ACAUCCGT 28 28nucleic acid single linear RNA Nucleotides at positions 1, 6-7, 10, 14,16-18, 20, 22, and 24-26 are 2′-fluoro (2′-F) modified Nucleotides atpositions 2-3, 8-9, 11-13, 15, 19, 21, 23, and 27 are 2′-OMethyl(2′-OMe) modified T in position 28 is a 3′-3′-linked deoxythymidine 6CGGAAUCAGU GAAUGCUUAU ACAUCCGT 28 28 nucleic acid single linear RNANucleotides at positions 1, 6-7, 10, 14, 16-18, 20, 22, and 24-26 are2′-fluoro (2′-F) modified Nucleotides at positions 2-3, 8-9, 11-13, 15,19, 21, 23, and 27 are 2′-OMethyl (2′-OMe) modified T in position 28 isa 3′-3′-linked deoxythymidine 7 CGGAAUCAGU GAAUGCUUAU ACAUCCGT 28 28nucleic acid single linear RNA Nucleotides at positions 1, 6-7, 10, 14,16-18, 20, 22, and 24-26 are 2′-fluoro (2′-F) modified Nucleotides atpositions 2-3, 8-9, 11-13, 15, 19, 21, 23, and 27 are 2′-OMethyl(2′-OMe) modified T in position 28 is a 3′-3′-linked deoxythymidine 8CGGAAUCAGU GAAUGCUUAU ACAUCCGT 28 28 nucleic acid single linear RNANucleotides at positions 1, 6-7, 10, 14, 16-18, 20, 22, and 24-26 are2′-fluoro (2′-F) modified Nucleotides at positions 2-3, 8-9, 11-13, 15,19, 21, 23, and 27 are 2′-OMethyl (2′-OMe) modified T in position 28 isa 3′-3′-linked deoxythymidine 9 CGGAAUCAGU GAAUGCUUAU ACAUCCGT 28 28nucleic acid single linear RNA Nucleotides at positions 1, 6-7, 10, 14,16-18, 20, 22, and 24-26 are 2′-fluoro (2′-F) modified Nucleotides atpositions 2-3, 8-9, 11-13, 15, 19, 21, 23, and 27 are 2′-OMethyl(2′-OMe) modified T in position 28 is a 3′-3′-linked deoxythymidine 10CGGAAUCAGU GAAUGCUUAU ACAUCCGT 28 28 nucleic acid single linear RNANucleotides at positions 1, 6-7, 10, 14, 16-18, 20, 22, and 24-26 are2′-fluoro (2′-F) modified Nucleotides at positions 2-3, 8-9, 11-13, 15,19, 21, 23, and 27 are 2′-OMethyl (2′-OMe) modified T in position 28 isa 3′-3′-linked deoxythymidine 11 CGGAAUCAGU GAAUGCUUAU ACAUCCGT 28 28nucleic acid single linear RNA Nucleotides at positions 1, 6-7, 10, 14,16-18, 20, 22, and 24-26 are 2′-fluoro (2′-F) modified Nucleotides atpositions 2-3, 8-9, 11-13, 15, 19, 21, 23, and 27 are 2′-OMethyl(2′-OMe) modified T in position 28 is a 3′-3′-linked deoxythymidine 12CGGAAUCAGU GAAUGCUUAU ACAUCCGT 28 28 nucleic acid single linear RNANucleotides at positions 1, 3-4, 6, 8-9, 14-16, 18, 20, 24, and 26 are2′-fluoro (2′-F) modified Nucleotides at positions 5, 7, 10, 12-13, 17,19, 21-23, 25, and 27 are 2′-OMethyl (2′-OMe) modified T at position 28is a 3′-3′-linked deoxythymidine 13 CAUUACACCG AAGUUUACGU GAGUAUGT 28 61nucleic acid single linear RNA All pyrimidines are 2′-fluoro (2′-F)modified 14 GGGAGGACGA UGCGGNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNCAGAC 50GACUCGCCCG A 61 61 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified 15 GGGAGGACGA UGCGGAAGAA UUGGUCAUCG UCGUCUCCGCCUCCCCAGAC 50 GACUCGCCCG A 61 61 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 16 GGGAGGACGA UGCGGAAUACGGAAGAAUUG GAUACAUAUG CUCGUCAGAC 50 GACUCGCCCG A 61 61 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 17GGGAGGACGA UGCGGGAUAA CAGAAGAAUU GGUGAACAAC GUGGUCAGAC 50 GACUCGCCCG A61 61 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified 18 GGGAGGACGA UGCGGAUGAU CGCGUAGGAA GUAUUGGAAGGCCCUCAGAC 50 GACUCGCCCG A 61 61 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 19 GGGAGGACGA UGCGGCACUUUAGAAGAAUU GAAUUUCCCG CUGGUCAGAC 50 GACUCGCCCG A 61 61 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 20GGGAGGACGA UGCGGUAGGA AGAAUUGGAA GCGCAUUUUC CUCGYCAGAC 50 GACUCGCCCG A61 61 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified 21 GGGAGGACGA UGCGGCGGGA UUUUGGAAGA AUUGGAUAUUGGCCUCAGAC 50 GACUCGCCCG A 61 61 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 22 GGGAGGACGA UGCGGCGGYACUUUGGAAGA AUUGAAUUUC CCGCUCAGAC 50 GACUCGCCCG A 61 61 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 23GGGAGGACGA UGCGGAAGAA UUGGAUAUAU CGUUCACCCC CACCUCAGAC 50 GACUCGCCCG A61 61 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified 24 GGGAGGACGA UGCGGAAACG GAAGAAUUGG AUACGCAAGCACGUUCAGAC 50 GACUCGCCCG A 61 61 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 25 GGGAGGACGA UGCGGUAGGAAGUAUUGUAA GCGCCUCGUU UUCGCCAGAC 50 GACUCGCCCG A 61 61 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 26GGGAGGACGA UGCGGAGUUU UGGAAGAAUU GGAUGUUCCG AUCGUCAGAC 50 GACUCGCCCG A61 61 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified 27 GGGAGGACGA UGCGGAAGAA ACGGAAGAAU UGGAGACACGCUCGUCAGAC 50 GACUCGCCCG A 61 71 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 28 GGGAGGACGA UGCGGGAAGAAUUGAUGUUG UAUUGUCCUU CCGAUUUCCU 50 GCCGUCAGAC GACUCGCCCG A 71 70nucleic acid single linear RNA All pyrimidines are 2′-fluoro (2′-F)modified 29 GGGAGGACGA UGCGGACAGA AGAAUUGGGC UUCGCAUUAU CCUCUGUCAG 50CCGCCAGACG ACUCGCCCGA 70 71 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 30 GGGAGGACGA UGCGGUGAGAGAAACGGAAG AAUUGGAUAC GAUACUCAUC 50 GCGCUCAGAC GACUCGCCCG A 71 61nucleic acid single linear RNA All pyrimidines are 2′-fluoro (2′-F)modified 31 GGGAGGACGA UGCGGCUUAA GUUUUGGAAG AAUUGAAUAC UGGGUCAGAC 50GACUCGCCCG A 61 60 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified 32 GGGAGGACGA UGCGGUAACC AGUGGAAGAA UUGGCUGCUAUCCUCAGACG 50 ACUCGCCCGA 60 60 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 33 GGGAGGACGA UGCGGAACGGAAGAAUUGGA UACGUAGCAU GCGUCAGACG 50 ACUCGCCCGA 60 61 nucleic acid singlelinear RNA All pyrimidines are 2′-fluoro (2′-F) modified 34 GGGAGGACGAUGCGGCAGGA UUUUGGAAGA AUUGGAUAUU GGCCGCAGAC 50 GACUCGCCCG A 61 61nucleic acid single linear RNA All pyrimidines are 2′-fluoro (2′-F)modified 35 GGGAGGACGA UGCGGAAACG GAAGAAUUGG AUACCGCUAC GUGUUCAGAC 50GACUCGCCCG A 61 61 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified 36 GGGAGGACGA UGCGGAAGAA UUGAGCAUUC CUUCUCCUUGUGCCUCAGAC 50 GACUCGCCCG A 61 61 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 37 GGGAGGACGA UGCGGAGCUAACGGAAGAAU UGGAAACAAC CGCGUCAGAC 50 GACUCGCCCG A 61 61 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 38GGGAGGACGA UGCGGYGAAC CGAUGGAAUU UUUGGACGCU CGCCUCAGAC 50 GACUCGCCCG A61 61 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified 39 GGGAGGACGA UGCGGAYCAA CCGAUUGACG UUAUGGGACGCUGGUCAGAC 50 GACUCGCCCG A 61 61 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 40 GGGAGGACGA UGCGGUAACCGAUUGAACUU CUUGGACGCU ACCGUCAGAC 50 GACUCGCCCG A 61 61 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 41GGGAGGACGA UGCGGUAACC GAAUUGAAGU UAUUGGACGC UACCUCAGAC 50 GACUCGCCCG A61 71 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified 42 GGGAGGACGA UGCGGGAGCA GAACCGAUAG AAGAAUUGGACGCUCAGCUC 50 CGGGUCAGAC GACUCGCCCG A 71 71 nucleic acid single linearRNA All pyrimidines are 2′-fluoro (2′-F) modified 43 GGGAGGACGAUGCGGGUACC AGAAUGAGCA ACCGAAUGAA GAACUGGACG 50 CUGCUCAGAC GACUCGCCCG A71 71 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified 44 GGGAGGACGA UGCGGUGAAC CGAUGGAAUC GCUUGGACGCUCAUCGCACG 50 UUGCUCAGAC GACUCGCCCG A 71 61 nucleic acid single linearRNA All pyrimidines are 2′-fluoro (2′-F) modified 45 GGGAGGACGAUGCGGUCAAC CGGUUGAAUA UUUGGUCGCU GACCUCAGAC 50 GACUCGCCCG A 61 61nucleic acid single linear RNA All pyrimidines are 2′-fluoro (2′-F)modified 46 GGGAGGACGA UGCGGAACUA GUGAAUGCUU AUACGACCGU GUUGUCAGAC 50GACUCGCCCG A 61 60 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified 47 GGGAGGACGA UGCGGAUCAG UGAAUGCUUA UAGACCGCCUCCGUCAGACG 50 ACUCGCCCGA 60 61 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 48 GGGAGGACGA UGCGGAGAAUCAGUGAAUGC UUAUAAAUCU CGYGUCAGAC 50 GACUCGCCCG A 61 62 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 49GGGAGGACGA UGCGGAAUCA GUGAAUGCUU AUAGCUCCCG CGUCCUCAGA 50 CGACUCGCCC GA62 60 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified 50 GGGAGGACGA UGCGGAACCA GUGAAUGCUU AUAAGACUGCUCGUCAGACG 50 ACUCGCCCGA 60 61 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 51 GGGAGGACGA UGCGGAUCAGUGAAUGCUUA UAGACCGUAU UGCGUCAGAC 50 GACUCGCCCG A 61 61 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 52GGGAGGACGA UGCGGAGAAU CAGUGAAUGC UUAUAAACCU CGUGUCAGAC 50 GACUCGCCCG A61 61 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified 53 GGGAGGACGA UGCGGAAUCA GUGAAUGCUU AUAGCUCCGCGUGGUCAGAC 50 GACUCGCCCG A 61 61 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 54 GGGAGGACGA UGCGGACCAGUGAAUGCUUA UAAGCCCAUC GACCUCAGAC 50 GACUCGCCCG A 61 60 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 55GGGAGGACGA UGCGGCAGGG UGAAUGCCAA UGUACUUUUC GCGUCAGACG 50 ACUCGCCCGA 6061 nucleic acid single linear RNA All pyrimidines are 2′-fluoro (2′-F)modified 56 GGGAGGACGA UGCGGAAUCA GUGAAUGCUU AUAGCUCCAC GUCGUCAGAC 50GACUCGCCCG A 61 61 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified 57 GGGAGGACGA UGCGGAAUCA GUGAAUGCUU AUACAUCCGCUCGGUCAGAC 50 GACUCGCCCG A 61 61 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 58 GGGAGGACGA UGCGGGACUAGGUGAAUGCC AAUAUUCUUC UCCGUCAGAC 50 GACUCGCCCG A 61 30 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified N atposition 30 is a 3′-3′-linked deoxythymidine 59 GACGAUGCGG UAGGAAGAAUUGGAAGCGCN 30 28 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified 60 GACGAUGCGG UAGGAAGAAU UGGAAGCG 28 28nucleic acid single linear RNA All pyrimidines are 2′-fluoro (2′-F)modified C in position 28 is 2′-OH C 61 ACGAUGCGGU AGGAAGAAUU GGAAGCGC28 23 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified C in position 23 is 2′-OH C 62 GCGGUAGGAA GAAUUGGAAG CGC23 22 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified C in position 22 is 2′-OH C 63 CGGUAGGAAG AAUUGGAAGC GC22 22 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified N at position 22 is a 3′-3′-linked deoxythymidine 64GGUAGGAAGA AUUGGAAGCG CN 22 21 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified N at position 21 is a3′-3′-linked deoxythymidine 65 GUAGGAAGAA UUGGAAGCGCN 21 32 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified N atposition 32 is a 3′-3′-linked deoxythymidine 66 GGCGAACCGA UGGAAUUUUUGGACGCUCGC CN 32 29 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified C in position 29 is 2′-OH C 67 GCGAACCGAUGGAAUUUUUG GACGCUCGC 29 27 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 68 CGAACCGAUG GAAUUUUUGGACGCUCG 27 26 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified N at position 26 is a 3′-3′-linkeddeoxythymidine 69 GAACCGAUGG AAUUUUUGGA CGCUCN 26 24 nucleic acid singlelinear RNA All pyrimidines are 2′-fluoro (2′-F) modified N at position24 is a 3′-3′-linked deoxythymidine 70 AACCGAUGGA AUUUUUGGAC GCUN 24 22nucleic acid single linear RNA All pyrimidines are 2′-fluoro (2′-F)modified N at position 22 is a 3′-3′-linked deoxythymidine 71 ACCGAUGGAAUUUUUGGACG CN 22 30 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified N at position 30 is a 3′-3′-linkeddeoxythymidine 72 GCGGAAUCAG UGAAUGCUUA UACAUCCGCN 30 27 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 73CGGAAUCAGU GAAUGCUUAU ACAUCCG 27 25 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified C in position 25 is 2′-OH C 74GGAAUCAGUG AAUGCUUAUA CAUCC 25 24 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified N at position 24 is a3′-3′-linked deoxythymidine 75 GAAUCAGUGA AUGCUUAUAC AUCN 24 22 nucleicacid single linear RNA All pyrimidines are 2′-fluoro (2′-F) modified Nat position 22 is a 3′-3′-linked deoxythymidine 76 AAUCAGUGAA UGCUUAUACAUN 22 20 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified N at position 20 is a 3′-3′-linked deoxythymidine 77AUCAGUGAAU GCUUAUACAN 20 29 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified Purines are 2′-OMethyl(2′-OMe) modified G in position 10, A in position 12, and G in position22 are unmodified (i.e., 2′-OH) 78 GACGAUGCGG UAGGAAGAAU UGGAAGCGC 29 29nucleic acid single linear RNA All pyrimidines are 2′-fluoro (2′-F)modified Purines are 2′-OMethyl (2′-OMe) modified G in position 10 and Ain position 12 are unmodified (i.e., 2′-OH) 79 GACGAUGCGG UAGGAAGAAUUGGAAGCGC 29 29 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified Purines are 2′-OMethyl (2′-OMe) modified G inpositions 10 and 22 is unmodified (i.e., 2′-OH) 80 GACGAUGCGG UAGGAAGAAUUGGAAGCGC 29 29 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified Purines are 2′-OMethyl (2′-OMe) modified A inposition 12 and G in position 22 are unmodified (i.e., 2′-OH) 81GACGAUGCGG UAGGAAGAAU UGGAAGCGC 29 31 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified Purines are 2′-OMethyl(2′-OMe) modified A in position 6 and G in position 21 are unmodified(i.e., 2′-OH) 82 GGCGAACCGA UGGAAUUUUU GGACGCUCGC C 31 31 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified Purinesare 2′-OMethyl (2′-OMe) modified A in position 6 is unmodified (i.e.,2′- OH) 83 GGCGAACCGA UGGAAUUUUU GGACGCUCGC C 31 31 nucleic acid singlelinear RNA All pyrimidines are 2′-fluoro (2′-F) modified Purines are2′-OMethyl (2′-OMe) modified G in position 21 is unmodified (i.e., 2′-OH) 84 GGCGAACCGA UGGAAUUUUU GGACGCUCGC C 31 29 nucleic acid singlelinear RNA All pyrimidines are 2′-fluoro (2′-F) modified Purines are2′-OMethyl (2′-OMe) modified A in positions 5 and 6 is unmodified (i.e.,2′-OH) 85 GCGGAAUCAG UGAAUGCUUA UACAUCCGC 29 29 nucleic acid singlelinear RNA All pyrimidines are 2′-fluoro (2′-F) modified Purines are2′-OMethyl (2′-OMe) modified A in position 5 is unmodified (i.e., 2′-OH) 86 GCGGAAUCAG UGAAUGCUUA UACAUCCGC 29 29 nucleic acid single linearRNA All pyrimidines are 2′-fluoro (2′-F) modified Purines are 2′-OMethyl(2′-OMe) modified A in position 6 is unmodified (i.e., 2′- OH) 87GCGGAAUCAG UGAAUGCUUA UACAUCCGC 29 23 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified Purines are 2′-OMethyl(2′-OMe) modified G in position 4 and A in position 6 are unmodified(i.e., 2′-OH) 88 GCGGUAGGAA GAAUUGGAAG CGC 23 29 nucleic acid singlelinear RNA All pyrimidines are 2′-fluoro (2′-F) modified Purines are2′-OMethyl (2′-OMe) modified A in position 5 and G in position 20 areunmodified (i.e., 2′-OH) 89 GCGAACCGAU GGAAUUUUUG GACGCUCGC 29 27nucleic acid single linear RNA All pyrimidines are 2′-fluoro (2′-F)modified Purines are 2′-OMethyl (2′-OMe) modified A in positions 4 and 5is unmodified (i.e., 2′-OH) 90 CGGAAUCAGU GAAUGCUUAU ACAUCCG 27 60nucleic acid single linear RNA All pyrimidines are 2′-fluoro (2′-F)modified 91 GGGAGGACGA UGCGGUCUUU GAGUUUUUGC CAACGGUUUU CGCUCAGACG 50ACUCGCCCGA 60 61 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified 92 GGGAGGACGA UGCGGAACGG AAUUCUUGGA UACACACCUCGUCCUCAGAC 50 GACUCGCCCG A 61 61 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 93 GGGAGGACGA UGCGGUCAGGAACGGAAUUU UUGGAGACAC GCCCUCAGAC 50 GACUCGCCCG A 61 61 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 94GGGAGGACGA UGCGGACUGG GAGAAUCCGA AAAACCUUCA CGCGUCAGAC 50 GACUCGCCCG A61 61 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified 95 GGGAGGACGA UGCGGAUCCA UCAUUUAACC GUUUGCUCUCCCCCUCAGAC 50 GACUCGCCCG A 61 61 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 96 GGGAGGACGA UGCGGUUGAUCGGACGUUAG UCAUUUCCCG AUCGUCAGAC 50 GACUCGCCCG A 61 61 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 97GGGAGGACGA UGCGGGAGCU UGAAGUUUCA GUAUUGGCAC AACCUCAGAC 50 GACUCGCCCG A61 61 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified 98 GGGAGGACGA UGCGGCGCCA CUUUGGAAGU UAUUGAAUUUCGCGUCAGAC 50 GACUCGCCCG A 61 61 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 99 GGGAGGACGA UGCGGUGAAUGAGCUGACGA CCCUGAAUUG CUCGUCAGAC 50 GACUCGCCCG A 61 61 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 100GGGAGGACGA UGCGGGAGCU UGAAGUUUCG GUAUUGGCAC AACCUCAGAC 50 GACUCGCCCG A61 61 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified 101 GGGAGGACGA UGCGGCAACU AUUCGUUGAU GUUUCCGUGAGCCGUCAGAC 50 GACUCGCCCG A 61 61 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 102 GGGAGGACGA UGCGGGAGCUUGAAGUUUCA GUACUGGCAC AACCUCAGAC 50 GACUCGCCCG A 61 61 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 103GGGAGGACGA UGCGGAACCA AUAGAGAUCU UCGGCUGCCC CGCGUCAGAC 50 GACUCGCCCG A61 61 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified 104 GGGAGGACGA UGCGGAAAAC GCUUUUCUUG GCCCCCUCGUUGCGCCAGAC 50 GACUCGCCCG A 61 61 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 105 GGGAGGACGA UGCGGUUAACGGAAUUCUUG GAUACAUAGC AUGGUCAGAC 50 GACUCGCCCG A 61 70 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 106GGGAGGACGA UGCGGCAAAG UUUGAGUUGA UCUGAUACGU UUCAGUAUUG 50 GCGUCAGACGACUCGCCCGA 70 71 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified 107 GGGAGGACGA UGCGGAUCUG UGAACUGGGUUUUUGCCGAC GGUUACGCUU 50 UUGCUCAGAC GACUCGCCCG A 71 71 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 108GGGAGGACGA UGCGGCAAAA GUUUGAGUUG AUCUGAUACG UUUCAGUAUU 50 GGCGUCAGACGACUCGCCCG A 71 71 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified 109 GGGAGGACGA UGCGGUUGAU CGAGGUUCUAAAGCCUAUUU CCUGACUUUC 50 UCCCCCAGAC GACUCGCCCG A 71 70 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 110GGGAGGACGA UGCGGAUCUG UGAACUGGGU UUUGCCGACG GUUACGCUUU 50 UGCUCAGACGACUCGCCCGA 70 71 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified 111 GGGAGGACGA UGCGGAAGGA AGAUGUUGAUCGUUUGACGU GAUGUGGAUC 50 CGCGUCAGAC GACUCGCCCG A 71 71 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 112GGGAGGACGA UGCGGUAGUA AGUUAUUGAA AGCGCAUCUC UAUCAACUCU 50 CGGCCCAGACGACUCGCCCG A 71 71 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified 113 GGGAGGACGA UGCGGUACUU UCUUCUUUCUUUGCCUUUCU UUUUCUUUUA 50 CGCCUCAGAC GACUCGCCCG A 71 71 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 114GGGAGGACGA UGCGGCAGUU AAUUAAUUUG AGUUGUGAUG UGUGUCGUUA 50 UGGGUCAGACGACUCGCCCG A 71 71 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified 115 GGGAGGACGA UGCGGGAUGC UGAGUGAGGAAGUCUGAUUG UUGCAGUAUU 50 GGCGUCAGAC GACUCGCCCG A 71 70 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 116GGGAGGACGA UGCGGAAUGG AAUUUGAGUC GAUCUAGAAU GCGUCGUAUG 50 GGCUCAGACGACUCGCCCGA 70 71 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified 117 GGGAGGACGA UGCGGACUCA ACUGGACGCUAUGUUGACGG UUAUCGCUUU 50 UGGGUCAGAC GACUCGCCCG A 71 71 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 118GGGAGGACGA UGCGGCAGGU UCAGAAUUGG CAGUCGCAUU GAUCUUUUUC 50 ACCGCCAGACGACUCGCCCG A 71 72 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified 119 GGGAGGACGA UGCGGCAAAA GUUUGAGUUGAUCUGAUACG UUUCCAGUAU 50 UGGCGUCAGA CGACUCGCCC GA 72 71 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 120GGGAGGACGA UGCGGCAGUU AAUUAACUUG AGUUGUGAUG UGUGUCGUUA 50 UGGGUCAGACGACUCGCCCG A 71 71 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified 121 GGGAGGACGA UGCGGCAAAU UCAAGGUCGAGUUAUGCGUA GAUGUGGCUC 50 CUGUGCAGAC GACUCGCCCG A 71 71 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 122GGGAGGACGA UGCGGAUCUG UUGAACUGGG UUUUGCCGAC GGUUACGCUU 50 UUGCUCAGACGACUCGCCCG A 71 71 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified 123 GGGAGGACGA UGCGGCAGUU AAUUAAUUUGAGUUGUGAUG UGUGUCGUUA 50 UGGGCCAGAC GACUCGCCCG A 71 61 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 124GGGAGGACGA UGCGGUUGAU CGAUUUUCCU GGCGUCCUUA UGGGUCAGAC 50 GACUCGCCCG A61 60 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified 125 GGGAGGACGA UGCGGUCUUU GGGUUUUUGC CAACGGUUUUCGCUCAGACG 50 ACUCGCCCGA 60 61 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 126 GGGAGGACGA UGCGGUUCAGAAUUGGCUGC GAUCCUUUUC CCCCUCAGAC 50 GACUCGCCCG A 61 61 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 127GGGAGGACGA UGCGGUUGAU CGACUUUUCC UGAUCUUCUC CUCCUCAGAC 50 GACUCGCCCG A61 61 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified 128 GGGAGGACGA UGCGGCACUA GGUGCAUGCC AUGAAAUCUUGCUGUCAGAC 50 GACUCGCCCG A 61 61 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 129 GGGAGGACGA UGCGGGAUCACGGCUUUGCA CGAUCUUCUU CUCCUCAGAC 50 GACUCGCCCG A 61 61 nucleic acidsingle linear RNA All pyrimidines are 2′-fluoro (2′-F) modified 130GGGAGGACGA UGCGGGAUCA CGAUACUUGA CGAUUUUCCU CUCCUCAGAC 50 GACUCGCCCG A61 61 nucleic acid single linear RNA All pyrimidines are 2′-fluoro(2′-F) modified 131 GGGAGGACGA UGCGGAGCGG UAUUCUGUUC GGUCGUUUUCCUCCUCAGAC 50 GACUCGCCCG A 61 61 nucleic acid single linear RNA Allpyrimidines are 2′-fluoro (2′-F) modified 132 GGGAGGACGA UGCGGAUUUGGAUGCAUGUC AAGGCGUUUU GCCCUCAGAC 50 GACUCGCCCG A 61 78 nucleic acidsingle linear DNA 133 TAATACGACT CACTATAGGG AGGACGATGC GGNNNNNNNNNNNNNNNNNN 50 NNNNNNNNNN NNCAGACGAC TCGCCCGA 78 88 nucleic acid singlelinear DNA 134 TAATACGACT CACTATAGGG AGGACGATGC GGNNNNNNNN NNNNNNNNNN 50NNNNNNNNNN NNNNNNNNNN NNCAGACGAC TCGCCCGA 88 16 nucleic acid singlelinear DNA 135 TCGGGCGAGT CGTCTG 16 32 nucleic acid single linear DNA136 TAATACGACT CACTATAGGG AGGACGATGC GG 32 13 nucleic acid single linearDNA 137 GCCTTAGTCA CTT 13 14 nucleic acid single linear DNA 138CGGATGTATA AGCA 14 71 nucleic acid single linear RNA All pyrimidines are2′-fluoro (2′-F) modified 139 GGGAGGACGA UGCGGNNNNN NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN 50 NNNNNCAGAC GACUCGCCCG A 71

We claim:
 1. A method for treating a VEGF mediated disease or medicalcondition selected from the group consisting of cancer, psoriasis,ocular disorders characterized by excessive angiogenesis, collagenvascular diseases and rheumatoid arthritis comprising administering apharmaceutically effective amount of a Complex comprised of a VEGFNucleic Acid Ligand comprising 2′F-modified nucleotides and aNon-Immunogenic, High Molecular Weight Compound or a Lipophilic Compoundto a patient in need of said Complex.
 2. The method of claim 1 whereinsaid Non-Immunogenic, High Molecular Weight Compound is a PolyalkyleneGlycol.
 3. The method of claim 2 wherein said Polyalkylene Glycol ispolyethylene glycol.
 4. The method of claim 3 wherein said polyethyleneglycol has a molecular weight of about between 10-80K.
 5. The method ofclaim 3 wherein said polyethylene glycol has a molecular weight of about20-45K.
 6. The method of claim 5 wherein said Complex has the structure

LigandComponent=fCmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfUfUmAfUmAfCmAfUfCfCmG-3′3′-dT(VEGF ligand).
 7. The method of claim 1 wherein said Lipophilic Compoundis a glycerol lipid.
 8. The method of claim 7 wherein said Complex isfurther associated with a lipid construct.
 9. The method of claim 8wherein said Complex has the structure

LigandComponent=fCmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfUfUmAfUmAfCmAfUfCfCmG-3′3′-dT(VEGF ligand).
 10. A method for improving the pharmacokinetic propertiesof a VEGF Nucleic Acid Ligand comprising 2′F-modified nucleotidescomprising: covalently linking said VEGF Nucleic Acid Ligand with aNon-Immunogenic, High Molecular Weight Compound or Lipophilic Compoundto form a Complex comprised of a VEGF Nucleic Acid Ligand and aNon-Immunogenic, High Molecular Weight Compound or Lipophilic Compound;and administering said Complex to a patient, whereby the pharmacokineticproperties of said VEGF Nucleic Acid Ligand are improved.
 11. A methodfor targeting a therapeutic or diagnostic agent to a specificpredetermined biological target that is expressing VEGF in a patientcomprising: covalently linking said therapeutic or diagnostic agent witha Complex comprised of a VEGF Nucleic Acid Ligand comprising2′F-modified nucleotides and a Non-Immunogenic, High Molecular WeightCompound or Lipophilic Compound, and administering said Complex to saidpatient, whereby said therapeutic or diagnostic agent is targeted to aspecific predetermined biological target that is expressing VEGF.
 12. Amethod of inhibiting VEGF mediated angiogenesis in a patient comprising:covalently linking a VEGF Nucleic Acid Ligand comprising 2′F-modifiednucleotides to a Non-Immunogenic, High Molecular Weight Compound orLipophilic Compound to form a Complex, said Complex being capable ofinhibiting VEGF mediated angiogenesis; and administering said Complex tosaid patient, whereby said VEGF mediated angiogenesis is inhibited. 13.A method of inhibiting the growth of tumors in a patient comprising:covalently linking a VEGF Nucleic Acid Ligand comprising 2′F-modifiednucleotides to a Non-Immunogenic, High Molecular Weight Compound orLipophilic Compound to form a Complex, said Complex being capable ofinhibiting the growth of tumors; and administering said Complex to saidpatient, whereby said growth of tumors is inhibited.
 14. The method ofclaim 13 wherein said tumors or cells or tissues surrounding said tumorsare expressing VEGF.
 15. The method of claim 13 wherein said tumors orcells or tissues surrounding said tumors are expressing VEGF receptors.16. The method of claim 15 wherein said tumors are comprised of Kaposi'ssarcoma cells.
 17. A method of inhibiting macular degeneration in apatient comprising: covalently linking a VEGF Nucleic Acid Ligandcomprising 2′ F-modified nucleotides to a Non-Immunogenic, HighMolecular Weight Compound or Lipophilic Compound to form a Complex, saidComplex being capable of inhibiting macular degeneration; andadministering said Complex to said patient, whereby said maculardegeneration is inhibited.
 18. The method of claim 17 wherein saidNon-Immunogenic, High Molecular Weight Compound is a polyalkyleneglycol.
 19. The method of claim 18 wherein said polyalkylene glycol ispolyethylene glycol.
 20. The method of claim 19 wherein saidpolyethylene glycol has a molecular weight of about between 10-80k. 21.The method of claim 19 wherein said polyethylene glycol has a molecularweight of about between 20-45k.
 22. The method of claim 21 wherein saidcomplex has the structure

LigandComponent=fCmGmGrArAfUfCmAmGfUmGmAmAfUmGfCfUfUmAfUmAfCmAfUfCfCmG-3′3′-dT(VEGF ligand).
 23. A method for prolonging the residence time of aNucleic Acid Ligand in an ocular application comprising attaching aNon-Immunogenic, High Molecular Weight Compound to a Nucleic Acid Ligandto form a Complex, and administering said complex to the eye, wherebythe residence time of said Nucleic Acid Ligand is prolonged.